Genetic and autoimmune modulators of brain function in neuropsychiatric illness and health
Dissertation
for the award of the degree
‘Doctor of Philosophy’ (Ph.D.)
Division of Mathematics and Natural Sciences
of the Georg‐August‐Universität Göttingen
within the doctoral program Center for Systems Neuroscience
of the Georg-August University School of Science (GAUSS)
submitted by
Bárbara Oliveira
born in Vila Nova de Famalicão, Portugal
Göttingen 2018
Doctoral thesis committee:
Prof. Dr. Dr. Hannelore Ehrenreich (1st referee) Clinical Neuroscience Max Planck Institute of Experimental Medicine
Prof. Dr. Wolfram-Hubertus Zimmermann (2nd referee) Institute of Pharmacology University Medical Center Göttingen
Prof. Dr. Nils Brose Department of Molecular Neurobiology Max Planck Institute of Experimental Medicine
Dr. Sonja Wojcik Department of Molecular Neurobiology Max Planck Institute of Experimental Medicine
Members of the examination board:
Prof. Dr. Jürgen Wienands Institute for Cellular and Molecular Immunology University Medical Center Göttingen
Prof. Dr. Klaus-Armin Nave Department of Neurogenetics Max Planck Institute of Experimental Medicine
Prof. Dr. Ralf Heinrich
Department of Neurobiology Institute for Zoology and Anthropology
Date of oral examination: 17.04.2018
Declaration
I hereby declare that the thesis “Genetic and autoimmune modulators of brain function in neuropsychiatric illness and health” has been written independently and with no other sources and aids than quoted.
Bárbara Oliveira Göttingen, 02.03.2018
ACKNOWLEDGEMENT
“The value of things is not the time they last, but the intensity with which they occur. That is why there are unforgettable moments and unique people!” Fernando Pessoa
Going through my thoughts of what the last four years have been, I cannot feel anything but gratitude. All the good and bad moments, the tears and laughter and the “I will never finish my PhD” breakdowns come together to put a smile on my face. I have been blessed with incredible people that shared this journey with me and to whom I will never be able to thank enough for so many unforgettable moments!
I start with the “Captain” ;) my supervisor, Prof. Hannelore Ehrenreich. You gave me the opportunity of joining an incredible team and to be a part of several interesting projects. I am sincerely grateful for all the challenges and learning experiences you provided me with. I realize now, that even in the moments when we disagreed, you helped me to become a more confident and outspoken person. I will always be grateful for the trust you had in me and the scientific guidance during my PhD.
I would like to thank my thesis committee members, Prof. Wolfram-Hubertus Zimmermann, Prof. Nils Brose and Dr. Sonja Wojcik for their valuable guidance and scientific input during my PhD. Your inquisitive minds challenged me to be and to do better! Thank you also to my examination board members, Prof. Jürgen Wienands, Prof. Klaus-Armin Nave and Prof. Ralf Heinrich for taking the time to consider my work.
Moreover, I would like to express my gratitude to all the people with whom I collaborated during my PhD; The Bochum team: Prof. Michael Hollmann, Daniel and Christina for all the fun data discussions over the phone; To Krasimira, Iris and Daria for your priceless help in introducing me to stem cell culture; To my dear Patapia, which was always ready to help and cheer me up, I would not have made it without you! And finally, to Prof. Silvio Rizzoli and Eugenio for your guidance and great scientific discussions during my calcium imaging experiments. A special thank you to Eugenio, for making me believe that everything would work out. You always had time for me, even when you did not have any time left.
My lovely colleagues at Clinical Neuroscience (past and present), you made this journey a hell of a ride! Every single one of you provided me with sweet memories that I will forever cherish. I have been more than lucky to be a part of such a nice team. I truly enjoyed our lunch and coffee breaks and all the scientific discussions over the last years. A special thank you to Nadine, the best technical assistant ever, for putting all of your heart in our cultures. It was amazing to work with you.
A special thank you to Martin, Julia and Michelle for sharing the "PhD representative adventure" with me: I enjoyed all the crazy moments we lived together, the pancakes marathon…the stress of organizing the retreat and the summer party… even the abstract book drama seems funny now ;)
The amazing people that I am lucky to have as friends, that always supported and encouraged me:
To the amazing women, without whom I would not have come to Göttingen: Isabel, Ines, Liliana and Catarina. Your support gave me the courage necessary to make a change and for that I will be forever grateful.
My Extrabrain colleagues: being a part of this program with such an amazing group of people was one of the best experiences of my life… all the meetings around Europe and the time we spent together were truly special!
To Olaf and Gwen for always pulling me higher... Literally! Our climbing sessions helped me to keep my sanity during these four years. Your positive energy was a miracle worker.
To Umer, for always being so attentive and kind to me, and off course thank you for all the delicious food!
Anja, my sweet Anja! I will miss your positive energy! And your muffins ;) You are the sunshine of the office.
Fernandella! Thank you for being such a kind person! I am so lucky to have as a friend!
Bekir, the Wolf, my antibody dealer. Thank you for always being ready to listen.
Beate, we had to become friends after bumping into each other in pretty much every GGNB course we attended You are one of the gifts that Göttingen provided me with.
Vikas, I don’t know anyone else with such a positive attitude in life! Spending time with you is always good for my immune system
Ludo, thank you for your random acts of friendship: the countless bounties and pringles, the mushroom risotto for dinner and for keeping me company in the late nights of working in the lab. You always put up with all the pranks kindly and most importantly without killing me. Thank you for teaching me how to dance, I am aware of how big the challenge was ;) not as big as having German class together though!
Giulia, Marina, Mara, Edda and Livia…my girls! Life in Göttingen would not be the same without you…As we once sang in the back of a taxi in a cold November night: I will always love youuuuuuuuu! We shared so many adventures… from Copenhagen to Barcelona, Rome, Basel, Leiden, Berlin, Edinburgh, Amsterdam, Oslo and so many more! Mara visiting you was always amazing! And that picture with Santa! OMG! Your thesis writing survival kit brought me so much joy! Edda, my dear…Göttingen is so small that we even had the same stalker And Livia…that weekend in the Vatican with Francesco, zucchini flowers and cacio pepe forever!!!
As for the other two ladies… my “work wives”:
Giulia, my constant source of entertainment and support. Thank you for turning the bad moments into a good laugh and for being such a good partner in crime! Great minds think alike, evil minds work together . You were always ready to cross Europe and live new adventures. Thank you for Columbus and after Columbus…for singing Edelweiss on the train in Salzburg, for the best walking tour ever in Edinburgh! And for all the silly things we did together like planting tulips in July. Most of all, thank you for your believe in me.
Marina, my life guru! You always push me to be better and do better… even things that I did not think I was able to do like participating in the Great Barrier Run. In the past years I had a lot of muscle pain to blame you for! You are my rock, and in the difficult moments you are always there to support me and make me believe in myself. May we always celebrate surviving the tough moments with a glass of red wine on a roof top thank you for the shitake burgers, the pouched eggs and all the cookie sessions! Your positive energy is contagious and I am incredible lucky to be your friend!
Joana and Ginie, my examples of women’s strength and power! You inspire me always! I am super lucky to have you in my life.
César and Monique, the best flat mates ever! You are family! And despite of the distance you are always present in the most important moments!
Finally, my family: the source of all my strength and inspiration in life! Filipe (my beloved George) and Adolfo, you thought me the wonders of being different. It is for you and because of you that every day I try to be a better researcher. Papi, Miquinhas & Diogo. None of this or whatever I have accomplished in life would be possible without your love and support. Your constant care and attention, despite the distance, are my driving force! Papi, you proved me that love is timeless, and every day I feel your support and encouragement to be better. You taught me that it is up to me to set my own limits. Miquinhas, my cheerleader! It is overwhelming to feel your love and belief in me. You are always the first one to say that I can do it! Diogo, you are always there when I need you, and growing up together made me a stronger and more independent person. All I am today, I owe to the three of you!
So, lovely people! For all we lived so far and the many amazing moments ahead of us I am deeply thankful!
To Papi, my hero.
“I am nothing.
I'll never be anything.
Apart from that, I have in me all the dreams in the world.”
Fernando Pessoa
TABLE OF CONTENTS
1. Introduction ...... 15
Scope of the present work ...... 35
2. Establishing a cell culture system for translational studies ...... 39
3. PROJECT I – All naturally occurring autoantibodies against the NMDA receptor subunit GluN1 have pathogenic potential irrespective of epitope and immunoglobulin class ...... 47
Overview of Project I ...... 47
Original publication ...... 49
4. PROJECT II – Uncoupling the widespread occurrence of anti-NMDAR1 autoantibodies from neuropsychiatric disease in a novel autoimmune model ...... 63
Overview of Project II ...... 63
Original publication ...... 67
5. PROJECT III – Excitation-inhibition dysbalance as predictor of autistic phenotypes ..... 85
Overview of Project III ...... 85
Original publication ...... 87
6. Summary and Conclusions ...... 91
7. Bibliography ...... 97
8. Appendix ...... 113
Accepted co-author publications ...... 113
Manuscript under revision ...... 147
List of abbreviations ...... 161
Curriculum vitae ...... 163
List of publications ...... 165
1. INTRODUCTION
Brain development and function
The outstanding complexity of the human brain is the product of an evolutionary journey that started millions of years ago. The human connectome is a myriad of specialized neuronal cell types and its synaptic connections, which add up to assemble the neural circuits and networks (van den Heuvel et al. 2016). Much more than the sum of its individual components, the structural and functional connectivity of the brain is shaped by the unique interaction between an individual and its environment.
In its most simple definition, the human adult brain is composed by an average of 86.1 billion neurons and an approximately equal number of glial cells with a heterogeneous distribution (Azevedo et al. 2009, Herculano-Houzel 2009). To achieve such numbers, tightly regulated molecular and cellular processes take place, starting from early stages in embryonic development until adulthood. In the ectoderm, undifferentiated cells are recruited to give rise to neural stem cells. Once in the neural plate, they acquire their identity and begin the first steps of differentiation to neuronal and glial lineages. A coordinated spatial and temporal regulation of gene expression takes place along with extensive cell proliferation. The default mode of neural induction proposes that, in the embryonic ectoderm cells adopt a neural fate as a result of inhibition of the bone morphogenic protein (BMP) (Kandel et al. 2000, Munoz- Sanjuan et al. 2002). The balance between self-renewal and differentiation allows neural stem cells to either remain in a multipotent status or develop to neural progenitor cells and, ultimately, to a mature progeny. Notch signalling is highly involved in controlling the balance between expansion of neural stem cells/neural progenitor cells and neural differentiation, by blocking neuronal differentiation and maintaining neural stem cells in an undifferentiated state (Kandel et al. 2000).
After neuroectoderm induction, neurulation takes place to form the neural tube. Cellular diversity in the central nervous system (CNS) depends on spatial patterning cues, responsible for production of different types of neural progenitor cells. Early neural induction and spatial patterning involves the transcription factors orthodenticle homeobox 1 (OTX1), LIM homeobox 1 (LIM1) and forkhead box protein A2 (FoxA2), and specify anterior neural tissue. Further refinement of anterior/posterior patterning is regulated by gradients of Wnts, and Wnt antagonism. Patterning of the telencephalon occurs along the dorsal-ventral axis by dorsally produced fibroblast growth factor 8 (FGF8), BMPs, and Wnts and by a ventral gradient of sonic hedgehog (Kandel et al. 2000, Germain et al. 2010).
The output of neural stem cells overtime is a dynamic process in which these cells either commit to a neuronal or glial cell fate. In the developing neocortex, cortical laminar organization is dictated by radial migration of neuronal progeny. In the ventricular and subventricular zones, expansion of the progenitor pool starts with several rounds of
17 INTRODUCTION
symmetric division of the neural stem cells. Later on, a small percentage of these cells undergo asymmetric cell divisions to generate the early born neurons. With the progression of neurogenesis, these earlier born neurons differentiate to radial glia cells, progenitor cells with the dual role of serving as a migratory scaffold for neurons and as neuronal progenitors themselves. Later-born neurons migrate past the earlier-born neurons, migrating radially to the cortical plate and resulting in six distinct cortical layers formed in an inside-out fashion. The end of the neurogenic period dictates a switch form neurogenesis to gliogenesis. Following the formation of the cortical layers, the radial glia cells in the ventricular zone lose competence to produce neurons and acquire competence to produce glia, terminally differentiating to astrocytes. Oligodendroglia originate in the anterior entopeduncular area of the telencephalon and later migrate tangentially to populate the cortex. The cerebral cortex is comprised primarily of glutamatergic projection neurons, originating from dorsal telencephalic progenitors, and of GABAergic interneurons of ventral origin that migrate tangentially from the ganglionic eminences into the cortical plate. Upon arriving to the cortical plate, both neuronal populations are instructed to stop migrating and proceed with differentiation, forming and extending dendrites and establishing synaptic connections (Schuurmans et al. 2002, Molyneaux et al. 2007, Germain et al. 2010, Kohwi et al. 2013, Silbereis et al. 2016).
In the cortex, glutamatergic and GABAergic neurons are responsible for information processing, with excitatory and inhibitory synaptic inputs being tightly coupled. In fact, interneurons inhibit glutamatergic cells and are excited by them. Excitatory glutamatergic signalling is mediated by a series of glutamate receptors, grouped in two main categories: the ianotropic or metabotropic receptors. The α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA), kainate (KA) and N-methyl-D-aspartate (NMDA) receptors represent the three main subtypes of ianotropic receptors. Inhibitory GABAergic signalling, triggered by γ-aminobutyric acid (GABA) is associated with type A GABA ianotropic
receptors (GABAAR) and the type B GABA metabotropic receptors (GABABR). Cortical transmission is largely mediated by ionotropic neurotransmitter receptors. Glutamate elicits excitation via the activation of AMPAR and NMDAR, while GABA evokes inhibition via
GABAAR (Kandel et al. 2000, Isaacson et al. 2011). Inhibition is somehow proportional to the excitation produced, resulting in a relatively constant excitation/inhibition (E/I) ratio that controls neural excitability. At the network level, balanced inhibition allows a progressive recruitment of firing neurons and prevents epileptiform discharges and excitotoxicity. Disruption of the E/I balance might impair brain function, and possibly contribute to neurological disorders such as autism and schizophrenia (Tao et al. 2014).
18 INTRODUCTION
NMDA receptors
NMDA receptors (NMDAR) are ionotropic glutamate-gated ion channels, assembled as heteromers in a tetrameric conformation, which are essential mediators of brain plasticity (Sheng et al. 1994, Paoletti et al. 2013). Their molecular composition is variable and usually associates two copies of the obligatory GluN1 subunit with two GluN2 subunits or a mixture of GluN2 and GluN3 subunits, resulting in different biophysical and pharmacological properties (Mayer 2011). Sequence homology within the seven subunits divides them into three subfamilies: GluN1 subunit, GluN2 subunits (GluN2A, GluN2B, GluN2C and GluN2D) and GluN3 subunits (GluN3A and GluN3B) (Paoletti 2011, Paoletti et al. 2013). Alternative splicing increases GluN1 variability and together with receptor subunit composition influences receptor properties such as ion conductance, affinity to agonistic agents and sensitivity to allosteric modulation, receptor desensitization characteristics and association with intracellular signalling molecules. Post-translational modifications influencing receptor function include glycosylation, palmitoylation, S-nitrosylation and phosphorylation (Paoletti et al. 2013, Lussier et al. 2015, Hogan-Cann et al. 2016, Iacobucci et al. 2017).
Each NMDAR subunit comprises four elements spanning the extracellular, transmembrane and intracellular regions (Figure 1). NMDAR extracellular epitopes sense diffusible ligands such as glutamate, glycine, H+, Zn2+ and respond by gating a Ca2+-rich cationic current. Flow of Na+, K+ and Ca2+ depends on the controlled gating of the transmembrane pore (Paoletti et al. 2013).
Figure 1. Structure of the GluN1 subunit of NMDAR. The extracellular amino-terminal domain (NTD) participates in allosteric regulation and subunit assembly; the ligand-binding domain (LBD; consisting of the S1 and S2 segments) is the binding site for glycine in GluN1 and glutamate in GluN2 or D-serine in GluN3 subunits. Three transmembrane helices (A, B and C) and a pore loop that contains the ion channel constitute the
19 INTRODUCTION
transmembrane domain (TMD). Finally, the intracellular carboxy-terminal domain (CTD) is involved in receptor trafficking, anchoring, and binding to downstream signalling molecules (Furukawa et al. 2005, Gielen et al. 2009, Mayer 2011, Paoletti 2011, Paoletti et al. 2013).
NMDAR activity is required for synaptogenesis, experience-dependent synaptic remodelling and long-term potentiation and depression (Lau et al. 2007, Paoletti et al. 2013). NMDAR subunit composition and number are not static. Different receptor subtypes coexist in the CNS depending on the developmental stage, cellular type, sub-cellular location and neuronal activity (Lau et al. 2007). Typically, they are found in the post-synapse in a di-heteromeric GluN1/GluN2A or tri-heteromeric GluN1/GluN2A/GluN2B conformation (Lau et al. 2007, Iacobucci et al. 2017). Peri-synaptic and extra-synaptic sites are enriched in GluN2B- containing receptors while at the synapse the GluN1/GluN2A conformation is more frequent. Enrichment in GluN1/GluN2A occurs upon a postnatal developmental switch in synaptic NMDAR phenotype from GluN2B to GluN2A (Lau et al. 2007, Gladding et al. 2011, Paoletti et al. 2013). Receptor number at the synapses is regulated by neuronal activity. While blocking neuronal activity promotes alternative ribonucleic acid (RNA) splicing and export of NMDAR from the endoplasmic reticulum to the synapse, receptor internalization and degradation through the ubiquitin–proteasome system is driven by chronic activity (Lau et al. 2007, Horak et al. 2014).
The presence of NMDAR and glutamate signalling goes beyond the CNS. NMDAR are expressed across a wide range of non-neuronal cells and tissues, including glial and endothelial cells, kidney, bone, pancreas, among others. Physiological tasks attributed to these non-neuronal receptors include bone deposition, wound healing, inhibition of insulin secretion, blood brain barrier (BBB) integrity and activity-dependent myelination (Skerry et al. 2001, Hogan-Cann et al. 2016).
NMDAR dysfunction is linked to synaptic defects and ultimately neurological and psychiatric conditions. Altered subunit expression, trafficking, localization or activity might underlie several phenotypes. These include neurodegenerative conditions such as Parkinson’s and Alzheimer’s disease in which glutamate toxicity contributes to neuronal loss (Mehta et al. 2013). Associated neuropsychiatric conditions include schizophrenia, anti-NMDAR encephalitis and autism spectrum disorders in which altered glutamate signalling due to either reduced or enhanced NMDAR function is implicated (Paoletti et al. 2013).
20 INTRODUCTION
The blood brain barrier
The BBB is a dynamic interface between the CNS and the blood. Together with the blood- cerebrospinal fluid (CSF) barrier, the blood-retinal barrier, the blood-nerve barrier and the blood-labyrinth barrier, the BBB exerts a bi-directional control of the molecular and cellular trafficking into the brain (Blanchette et al. 2015).
The development of the BBB starts with the vascularization of the neuroepithelium via sprouting angiogenesis and consequent invasion of the neural epithelium by endothelial progenitor cells and pericytes in a later stage (Hellstrom et al. 1999, Stenman et al. 2008, Engelhardt et al. 2014). The brain endothelial cells interact with neural, vascular and immune cells to regulate its permeability via intra and intercellular events (Neuwelt et al. 2008, Ransohoff 2009, Neuwelt et al. 2011). The extracellular matrix, located at the abluminal endothelial surface acts as central scaffold, linking different cells and molecules of the BBB and providing a physical barrier for leukocyte migration (Correale et al. 2009, Blanchette et al. 2015).
The maintenance of the BBB properties involves the interaction of brain endothelial cells with different cell types and environmental cues. On the luminal side, a paracellular barrier is created via tight junctions to seal the space between adjacent brain endothelial cells and prevent unspecific influx of ions and small charged molecules from the blood stream (Huber et al. 2001, Blanchette et al. 2015). The assembly of tight junctions requires the transmembrane proteins occludin, claudin and junctional adhesion molecules. On the abluminal side, astrocyte-derived signals regulate the BBB phenotype by preventing immune cell infiltration and sealing the paracellular space (sonic hedgehog) (Alvarez et al. 2011); decreasing vascular permeability (angiopoietin) (Lee et al. 2003); and regulating tight junction integrity (angiotensin and Apolipoprotein E; ApoE) (Wosik et al. 2007, Bell et al. 2012). ApoE immunoreactivity in the brain is evident in astrocytic end-feet (Boyles et al. 1985) (Figure 2A). There, it regulates tight junction integrity through the activation of protein kinase C and phosphorylation of occludin (Nishitsuji et al. 2011). In fact, ApoE-/- mice display extravasation of serum immunoglobulin G (IgG) in the cerebellum and discrete cortical and subcortical areas such as the hippocampus (Fullerton et al. 2001).
Homeostasis in the brain is kept by specific transport systems and enzymes in the BBB. While drug and nutrient-metabolizing enzymes process neuroactive blood-borne compounds, specific transport systems in the plasma membrane of brain endothelial cells allow the passage of nutrients and water-soluble compounds (Correale et al. 2009). This balance can be altered by inflammatory cytokines, hormones and drugs (Blanchette et al. 2015). During neuroinflammation, inflammatory cytokines in the CNS or blood, as interleukin-1β (IL-1β), tumour necrosis factor α (TNF-α), CC-chemokine ligand 2 (CCL-2), and interleukin-17A (IL-
21 INTRODUCTION
17A), modulate the BBB permeability by degrading tight junction proteins, modifying their phosphorylation status or affecting their turnover rate (Argaw et al. 2006, Stamatovic et al. 2009, Marchiando et al. 2010). Breakdown of the BBB allows a free flow of blood-derived components and neurotoxic proteins that can accumulate in the CNS and lead to neuronal toxicity and progressive neurodegeneration (Winkler 2012; Montagne 2015). Additionally, influx of immunoglobulins (Ig) through the brain can occur via modulation of barrier properties due to brain endothelial cells activation or by transfer of antibodies in the absence of brain endothelial cell activation. Efflux to the circulation of up taken antibodies is mediated by neonatal Fc receptors, present in brain endothelial cells which actively transport immunoglobulins out of the brain (Figure 2B) (Zhang et al. 2001). Additionally, the BBB is not a homogenous structure. There is a differential distribution of the receptors for the modulating molecules of BBB permeability that, ultimately, can mediate different effects of the same circulating Ig on brain function by determining its primary entry site (Brimberg et al. 2015).
Figure 2. BBB and Ig access to the brain parenchyma. (A) Basic cellular composition of the BBB. (B) Mechanisms of Ig influx to the brain: via endothelial cell activation due to interaction of pro-inflammatory molecules with Toll-like receptor 4 (TLR4) (a) or cytokine receptors (b); transcellular-dependent mechanisms can transport cytokines and chemokines through the BBB and activate CNS immune cells (c) or by direct binding of circulating antibodies (d). Transfer of antibodies in the absence of endothelial cell activation can occur by receptor-mediated endocytosis (e), retrograde axonal transport by neurons protruding towards the lumen of BBB capillaries (f) or by transendothelial migration of B cells (g). Neonatal Fc receptors present in endothelial cells mediate efflux of Ig back to the circulation (h) (Fabian et al. 1987, Roth et al. 2004, Ge et al. 2008, Diamond et al. 2009). ApoE: apolipoprotein E; LPS; lipopolysaccharide; ABs: autoantibodies; Fc: Fragment crystallisable.
22 INTRODUCTION
Synergies between nervous and immune systems
Both the CNS and the immune system are self-organizing: they start with genetically encoded primary instructions, to which information retrieved from environmental cues is added to develop individualized systems. Once seen as two unrelated systems, the bi- directional interaction between brain and immune molecules has been challenging this view.
Two major classes of immune effector molecules – cytokines and antibodies – have been linked to brain development and function (Boulanger 2009, Deverman et al. 2009, Brimberg et al. 2015). The BMPs, belonging to the transforming growth factor β (TGF-β) cytokine superfamily, regulate induction of the neuroepithelium and signalling via the gp130 cytokine family maintains the radial glia cells pool by promoting its self-renewal during embryogenesis (Hatta et al. 2002, Munoz-Sanjuan et al. 2002). Also chemokines, small cytokines with chemoattractant properties, are implicated in migration, proliferation and differentiation of neurons and glia. The stromal cell-derived factor 1 (SDF-1) chemokine and its receptor C-X- C chemokine receptor type 4 (CXCR4) regulate cell proliferation and migration in the brain (Lu et al. 2002, Stumm et al. 2003).
Microglia, the resident macrophage-like cells in the brain are responsible for immune surveillance, responding to infection and injury by secreting a large repertoire of cytokines and chemokines. They scan the brain parenchyma making transient contacts with synapses (Wake et al. 2009). During embryogenesis, microglia promotes astrocyte proliferation by secreting IL-1 and regulates developmental apoptosis and synaptogenesis by secreting TNF- α (Giulian et al. 1988, Deverman et al. 2009). Immature astrocytes induce the expression of the complement cascade protein C1q on retinogeniculate neurons. The complement system, besides their opsonizing functions plays a role in synaptic elimination. Indeed, C1q and C3 complement components localize at the synapses and tag unwanted synapses for elimination by microglia during synaptic pruning (Stevens et al. 2007, Schafer et al. 2012). The class I major histocompatibility complex (MHC) engages antigen presentation to T cells during adaptive immune responses. In the brain it modulates plasticity in the hippocampus and participates in synapse refinement processes (Huh et al. 2000, Bhat et al. 2009, Lee et al. 2014). Antibodies targeting brain proteins also impact brain function and homeostasis, and the effects of brain exposure will be discussed later in the “Neurological diseases driven by autoimmunity” section of the introduction.
The crosstalk between the nervous and immune systems is not unidirectional and CNS molecules also interact with immune cells in the periphery. Of particular interest is the role of neurotransmitter signalling in immune cells. In the CNS, GABA is the major neurotransmitter involved in inhibitory processes. Additionally, immune cells like macrophages, T cells and antigen presenting cells (APC) express functional GABAAR. GABA synthesis by
23 INTRODUCTION
macrophages, dendritic cells and T cells has immunoinhibitory effects as downregulation of CD4+ T cell-mediated autoimmune processes (Tian et al. 2004, Bhat et al. 2010, Dionisio et al. 2011). In dendritic cells, GABAergic signalling potentiates chemotactic responses by promoting hypermotility (Fuks et al. 2012, Barragan et al. 2015).
Glutamate is another player in neurotransmitter-driven immunomodulation extending its role beyond excitatory processes in the brain. After maturation in the thymus, resting T cells express several metabotropic glutamate receptors (mGluR): mGlu2/3R mGlu5R, mGlu8R and ionotropic receptors like NMDAR, AMPAR and KA receptors. Dendritic cells undergoing maturation and in contact with T cells release glutamate to prevent T cell activation (Levite 2008). T cells initially uptake glutamate via the constitutively expressed mGluR5. Upon antigen presentation by dendritic cells and T cell activation, mGluR1 are expressed for glutamate uptake and attenuate the mGluR5-triggered effects, mediating enhanced T cell proliferation and secretion of pro-inflammatory cytokines (Levite 2008). Hence, during cross talk between dendritic and T cells, glutamate signalling appears to control the proliferation of T cells depending on which receptor is involved on its uptake (Pacheco et al. 2006, Pacheco et al. 2007). Additionally, T cell responses seem to be modulated by glutamate signalling via NMDAR upregulation upon CD4+ T cell activation. T helper 1 versus T helper 2 cell functions such as proliferation, cytokine production and cell survival seem to be differentially affected by NMDAR signalling. In vitro, pharmacological stimulation of NMDAR results in reduced T helper 1-like cytokine production and unaltered T helper 2-like or IL-10 responses, most probably due to susceptibility of T helper 1 cells to NMDAR-dependent physiological cell death (Orihara et al. 2018). However, a direct link between glutamate and NMDAR signalling in immune cells has not been established yet.
Innate and adaptive immunity
Down the road of (auto)immunity there are two ways to go: the innate or the adaptive one (Figure 3). When a prompter response to danger is required, innate immunity takes place and immune events are driven by transmembrane receptors like Toll-like receptors, cytokine and chemokine receptors or fragment crystallisable (Fc) receptors (Figure 3A) (Alberts et al. 2002, Church et al. 2008, Bhat et al. 2009). On the other side, adaptive immunity involves antigen-specific T cells and an orchestrated antibody response by B cells to antigen-driven stimulation (Figure 3B). In the core of adaptive immune responses is the production of high affinity antibodies able to recognize virtually any antigen due to somatic hypermutation events of Ig genes (Bhat et al. 2009). Both innate and adaptive immunity can be involved in autoimmune events. While innate-related autoimmunity events are mainly associated with the inflammasome in adaptive-related autoimmunity, involvement of immune cells as
24 INTRODUCTION macrophages, T and B cells as well as production of antibodies recognizing self-antigens and activation of the complement system are possible (Church et al. 2008).
Antibody production is the result of the combined activity of innate and adaptive immunity. In fact, immunostimulants, secreted during innate responses can promote inflammation and trigger adaptive immune responses in which macrophages and dendritic cells can engage in antigen presentation to naïve T cells (Alberts et al. 2002). T cell receptors (TCR) recognize both peptide segments of antigenic proteins and fragments of the antigen-bound MHC on the surface of APCs (Figure 3B). If on one side naïve T cells interact with dendritic cells during its activation, B cells require stimulation via activated T helper cells (Alberts et al. 2002). In the germinal centres of the lymph nodes activated B cells can differentiate to plasma cells or memory B cells. Differentiation of B cells into antibody-synthetizing plasma cells allows mass production and secretion of specific antibodies. Post-proliferative plasma cells usually secrete IgM, IgG or IgA antibodies with moderate affinity. Further cycles of B cell proliferation, somatic hypermutation and affinity purification leads to differentiation to plasma cells able to secrete antibodies with increased affinity. Additionally, memory B cells can be re-stimulated during a second encounter with the antigen and engage secondary antibody responses (Janeway et al. 2001).
Antigen recognition leads to lymphocyte activation and clonal expansion, producing clones of lymphocytes carrying the same antigen-specific receptor (Rose 2015). Mechanisms of tolerance to self-antigens must take place to prevent severe autoimmune reactions. Immunological tolerance is acquired by clonal deletion and inactivation of developing lymphocytes. Thus, the mature lymphocyte repertoire is shaped by negative and positive selection. Self-tolerance is granted by elimination or neutralization of lymphocytes with strongly self-reactive receptors during negative selection. Positive selection identifies and preserves lymphocytes fit to respond to foreign antigens (Janeway et al. 2001).
Immunoglobulin diversification
Immunoglobulins are Y-shaped molecules presenting two distinct regions: the variable (V) region that controls the specificity to bind to the antigen and the constant (C) region that determines how the antigen is eliminated, upon binding by recruiting cells and immune molecules to destroy the antigen source via phagocytosis or the complement system (Figure 3C). Membrane bound Ig on B cell surface, serve as cell receptor (BCR) for the antigen and have no effector functions. Its V regions, exposed on the cell surface, recognize and bind to antigens in order to activate B cells, promote clonal expansion and the production of specific antibodies (Janeway et al. 2001).
25 INTRODUCTION
In early stages of B cell development, the primary antibody repertoire is created by assembly of exons encoding the antigen-binding variable regions of Ig heavy and light chains (Hwang et al. 2015). Diversification of Ig genes in mature B cells can occur via two deoxyribonucleic acid (DNA) modifying mechanisms: somatic hypermutation and class switch recombination (Kracker et al. 2011). Somatic hypermutation takes place in the germinal centres and enables the selection of antibodies with increased antigen affinity by introducing point mutations in the exons of Ig heavy and light chains (Rajewsky 1996). This stochastic process generates an extensive repertoire of immunoglobulins able to recognize virtually any antigen. To produce high affinity antibodies antigen-activated B cells undergo affinity maturation processes, including multiple rounds of somatic hypermutation and selection of clones with high antigen affinity followed by clonal expansion (Hwang et al. 2015). Class switch recombination modulates antibody’s effector function. Immature B cells express mainly IgM and, upon antigen stimulation and interaction with T cells in the periphery they proliferate, differentiate and acquire the ability to produce antibodies of other isotypes. During class switch recombination events, the coding exons of the C region, within the IgH gene, are replaced by DNA recombination between switch (S) region DNA segments, while the binding specificity (V region) of the BCR is maintained (Selsing 2006, Kracker et al. 2011).
In mammals there are five isotypes of antibodies: IgA, IgD, IgE, IgG, and IgM (Lefranc et al. 2001). In the bone marrow, the first isotype produced by a developing B cell is IgM, to be inserted in the plasma membrane as the BCR of immature naïve B cells. Upon migration to peripheral lymphoid organs, these cells start to express IgD at their surface evolving to mature naïve B cells responsive to foreign antigens. IgM is the major isotype secreted into the bloodstream upon first exposure to the antigen. In its secreted form, IgM is a pentameric molecule with a total of ten antigen-binding sites and the ability to activate the complement system, while IgD seem to function mostly as cell-surface receptors. IgG is a monomer heavily produced during secondary immune responses and the main isotype present in the blood. Besides activating the complement system, its Fc region can signal to phagocytic cells via Fc receptors present in macrophages and neutrophils and, ultimately, trigger phagocytosis. IgA is the main antibody isotype in body fluids. Present in the blood as a monomer, it acquires a dimeric conformation, when present in secretions, thought to be protective against the proteolytic action of enzymes. IgE molecules are monomers that serve as cell-surface receptors for antigen in mast cells and basophils. After being secreted, IgE bind to Fc receptors in mast cells and, upon binding to an antigen, it signals the mast cell to release histamine (Janeway et al. 2001, Lefranc et al. 2001, Alberts et al. 2002).
26 INTRODUCTION
Figure 3. Innate and adaptive immunity. (A) Innate responses are mediated by recognition of foreign molecules by transmembrane receptors located in antigen presenting cells (APCs). Stimulation of these receptors activates intracellular signalling pathways as Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) or nuclear factor kappa B (NF-ĸB) that culminate in triggering the inflammasome and its major player interleukin 1 (IL-1). (B) Adaptive responses rely partially in pre-existing elements of innate immunity for T cell activation. Näive T cells are able to bind to antigen-MHC complexes via its T cell receptor (TCR) and engage proliferation and
27 INTRODUCTION
differentiation to effector cells as regulatory (Tr), cytotoxic (Tc) and helper (Th) T cells. APCs secrete cytokines that influence the functional differentiation of T cells. CD4+ T cells differentiate to Th1 in the presence of IL-12 and IFN-ɣ, while secretion of IL-4 promotes Th2 differentiation. This has a direct impact on the outcome of the immune response as Th1 preferentially activate macrophages and Th2 activate B cells. Upon activation by Th2 cells in the germinal centres, antigen-stimulated B cells undergo proliferation and differentiation events that culminate in high affinity antibody production by plasma cells and acquisition of immunological memory. (C) Antibody structure. Four polypeptide chains: two identical light (L) chains and two identical heavy (H) chains connected by a combination of noncovalent and covalent disulphide bonds (Alberts et al. 2002). IFN-γ: interferon gamma; MHC: major histocompatibility complex; Fc: fragment crystallisable region; Fab: antibody-binding fragment.
Autoimmunity – a case of molecular misunderstanding?
An autoimmune condition occurs when a specific adaptive immune response develops against a self-antigen, leading to chronic inflammatory tissue damage. As in a protective immune response, self-antigen triggered T cytotoxic cell responses and activation of macrophages by T helper 1 can cause tissue damage, whereas inappropriate T helper 2- mediated activation of self-reactive B cells can initiate detrimental autoantibody responses. Although the T and B cell repertoire are purged of most self-antigen high-affinity receptors by clonal deletion, they still include low-affinity self-reactive receptors. Transient autoimmune responses are common and pathological status arises only when they are sustained and a cause of tissue damage. Susceptibility to autoimmune disease has been most consistently associated with human leukocyte antigen (HLA) gene complex. The T cell response to an antigen depends on the HLA haplotype. Being so, susceptibility to autoimmunity can be determined by different levels of efficacy of MHC variants, coded by the HLA complex, in presenting autoantigenic peptides to autoreactive T cells. Additionally, during the selection of the TCR repertoire, self-antigens associated with certain MHC variants might induce positive selection of cells bearing TCRs specific for these self-antigens. A scenario only possible if these self-antigens are expressed at low levels or bind too poorly to self-MHC molecules and do not trigger negative selection events (Janeway et al. 2001).
Autoimmune responses can be initiated by molecular mimicry events during immune responses to foreign antigens, possibly due to sequence homology between human and the pathogen peptides. According to the molecular mimicry hypothesis, autoreactive T cells and autoantibodies are initially directed to microbial antigens and further react with similar self- antigens (Fujinami et al. 1985, Ang et al. 2004, Atassi et al. 2008). Immunological cross- reactivity can occur due: (i) to homology between amino acid sequences, (ii) recognition of non-homologous peptide sequences by a single BCR or TCR due to their high level of degeneracy, (iii) variability in antigen recognition by T cells during antigen presentation by influence of HLA haplotypes and (iv) recognition of structural similarity in complex molecular
28 INTRODUCTION structures by immunological receptors, that might include double-stranded DNA molecules or glycolipids for example (Fujinami et al. 1985, Mason 1998, Ang et al. 2004). Being so, one can define molecular mimicry as a dual recognition of self and non-self-peptides by a single BCR or TCR, in which cross-reactive antibodies and T cells can engage autoimmune events (Ang et al. 2004). A role for molecular mimicry has been proposed in some immune- mediated diseases including acute rheumatic fever (Group A streptococci), Chagas’ disease (Trypanosoma cruzi) and Guillain–Barre’ syndrome (Campylobacter jejuni) as examples (Ang et al. 2004, Sheikh et al. 2004, Teixeira et al. 2011, Cunningham 2012).
Neurological diseases driven by autoimmunity
In several autoimmune conditions, the presence of autoantibodies can be a direct cause of the disease as it happens in systemic lupus erythematosus, myastenia gravis and Rasmussen encephalitis or it can contribute to the severity of the disease as in rheumatoid arthritis. When it comes to autoimmune responses to brain antigens, autoantibodies can induce brain damage and likely initiate or worsen multiple neurologic conditions (Brimberg et al. 2013, Mader et al. 2017). Their contribution to brain pathology is dependent on BBB function that usually prevents their access to the brain (Hammer et al. 2014, Platt et al. 2017). Brain-reactive autoantibodies can target neuronal or non-neuronal antigens. In neuropsychiatric systemic lupus erythematosus, autoantibodies targeting double stranded DNA molecules, can cross react with the GluN2A and GluN2B subunits of the NMDAR and induce excitotoxic neuronal death due to prolonged channel opening time and exacerbated calcium influx (DeGiorgio et al. 2001, Faust et al. 2010). In neuromyelitis optica autoantibodies target the astrocytic aquaporin 4 water channel present in astrocytic endfeet, resulting in astrocyte loss and demyelination (Lennon et al. 2004).
In utero exposure to certain brain-reactive autoantibodies can lead to neurodevelopmental changes in the fetal brain. Contactin-associated protein 2 (CASPR2) autoantibodies, isolated from seropositive mothers of autistic children, when injected to female mice lead to neuronal abnormalities and an autistic-like phenotype in their male offspring upon in utero exposure. These include abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as impairments in sociability, flexible learning and repetitive behaviour (Brimberg et al. 2013, Brimberg et al. 2016).
Two outcomes are possible upon entry of brain-reactive autoantibodies in the brain. Remission of the neuropsychiatric symptoms can be achieved by removal of the autoantibodies, as it is the case in limbic encephalitis, associated predominantly with antibodies targeting the leucine-rich glioma inactivated protein 1 (LGI1) (Mader et al. 2017).
29 INTRODUCTION
Alternatively, exposure to brain-reactive autoantibodies might have persistent effects by triggering irreversible mechanisms that cannot be reverted by autoantibodies elimination. In neuropsychiatric systemic lupus erythematosus patients, acute exposure to GluN2A/GluN2B autoantibodies leads to neuronal apoptosis and chronic damage of surviving neurons mediated by microglia-dependent synaptic loss persistent upon autoantibodies removal (DeGiorgio et al. 2001, Faust et al. 2010, Bialas et al. 2017).
Rasmussen’s encephalitis was one of the first autoimmune conditions to be associated with neuronal surface autoantibodies targeting the glutamate system (Rogers et al. 1994). Since the identification of the pathogenic role of GluR3 autoantibodies in this condition, several other CNS disorders have been related to autoimmune processes targeting ion channel and synaptic proteins in the brain (Table 1). Of those, anti-NMDAR encephalitis was one of the first synaptic autoimmune encephalitides to be characterized at the molecular level (Dalmau et al. 2007).
30 INTRODUCTION
Table 1. Antibodies targeting neuronal or synaptic proteins and associated disorders.
Target Antibody effects Associated disorder Phenotype Endocytosis of NMDAR in Psychosis, seizures, GluN1 neurons with disruption of epitope Anti-NMDAR encephalitis dyskinesia function AMPAR Endocytosis of AMPAR in neurons Limbic encephalitis Memory loss, confusion Blockade of induction of long-term depression in Purkinje cells Cerebellar ataxia, Reduction of basal mGluR1 Reduction of AMPAR clusters at limbic encephalitis activity of Purkinje cells the synapse Limbic encephalitis, mGluR5 Unknown Memory loss, confusion Ophelia syndrome
Reduction of GABAAR at the GABAAR Encephalitis Seizures synapse and extrasynaptic sites
GABABR Unknown Limbic encephalitis Memory loss, seizures Endocytosis of GlyRα1 in HEK GlyRα1 PERM Muscle rigidity, spasms cells Basal ganglia D2R Unknown encephalitis, Parkinsonism Tourette syndrome Decreased expression of Neurexin3α Neurexin3α on synapses and Encephalitis Seizures, confusion decreased synapse formation Alteration of gephyrin clusters in Morvan syndrome, Memory loss, sleep CASPR2 inhibitory synapses limbic encephalitis disorder, neuromyotonia Inhibition of interaction with ADAM22; Decrease of LGI1 Limbic encephalitis Memory loss, seizures postsynaptic AMPAR with disruption of epitope function Disruption of vesicle endocytosis Stiff-person Rigidity, spasms, Amphiphysin in neurons encephalomyelitis confusion, memory loss Decrease of cell surface IgLON5 Sleep apnea, brainstem IgLON5 Sleep disorder in neurons dysfunction Hyperexcitability of enteric PERM, cerebellar ataxia, Hyperekplexia, diarrhoea DPPX neurons encephalitis and other GI symptoms
Notes: GluN1: glutamate ionotropic receptor NMDA type subunit 1; AMPAR: α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptor; mGluR1/5: metabotropic glutamate receptor 1/5; GABAAR: γ-aminobutyric acid type A receptor; GABABR: γ-aminobutyric acid type B receptor; GlyRα1: glycine receptor α1 subunit; D2R: dopamine D2 dopamine receptor; CASPR2: contactin-associated protein 2; LGI1: leucine-rich glioma inactivated protein 1; IgLON5: immunoglobulin G superfamily member 5; DPPX: dipeptidyl aminopeptidase-like protein 6; NMDAR: N-methyl-D-aspartate receptor; HEK: human embryonic kidney; ADAM22: disintegrin and metalloproteinase domain-containing protein 22; PERM: progressive encephalitis with rigidity and myoclonus; GI: gastrointestinal (Diamond et al. 2013, Crisp et al. 2016, Dalmau 2016).
31 INTRODUCTION
Anti-NMDAR encephalitis
Anti-NMDAR encephalitis patients develop psychosis, cognitive problems and seizures, and its clinical picture can progress to altered status of consciousness, dyskinesias and autonomic dysfunction (Dalmau et al. 2007). At the cellular level, binding of GluN1 autoantibodies (NMDAR-AB) leads to a reduction of NMDAR cluster density, mediated by direct disruption of the epitope upon binding, receptor internalization and degradation (Hughes et al. 2010, Gleichman et al. 2012). This decrease in NMDAR has been reported in vitro using mouse or rat hippocampal neurons exposed to CSF or IgG extracts of anti- NMDAR encephalitis patients or individuals with other conditions (Hughes et al. 2010, Hammer et al. 2014, Moscato et al. 2014). Additionally, surface and total GluN2A and GluN2B protein levels which assemble with GluN1 to form functional NMDARs, decrease upon exposure to patients autoantibodies (Hughes et al. 2010).
In the context of anti-NMDAR encephalitis, only IgG antibodies have been reported to bind to the extracellular NTD of the GluN1 subunit. The NTD domain includes seven N-linked consensus glycosylation sites (G1-G7); and glycosylation and deamidation of the G7 site contribute to epitope formation and recognition by NMDAR-AB (Gleichman et al. 2012). Exposure to patient-derived CSF prolongs the duration of NMDAR opening in HEK293 cells expressing GluN1-GluN2B, and induces reduction of long-term potentiation in acute hippocampal slices (Gleichman et al. 2012, Zhang et al. 2012, Jezequel et al. 2017). Moreover, injection of IgG or CSF into rodent brains leads to increased glutamate levels and excitability of the motor cortex (Manto et al. 2010, 2011). In vitro, NMDAR-AB have no binding preference to inhibitory or excitatory neurons (Moscato et al. 2014). Thus, in theory, the acute effects of antibody exposure, such as seizures might be due to a hyper- glutamatergic state and consequent increase in network excitability coupled with homeostatic changes in inhibitory neurotransmission (Crisp et al. 2016). Although NMDAR-AB do not induce compensatory changes in glutamate receptor gene expression upon receptor internalization in vitro, they cause a decrease in inhibitory synapse density onto excitatory
hippocampal neurons. In fact, a reduction on GABAAR cluster density has been observed in + hippocampal neurons exposed to NMDAR-AB CSF with no change in GABAAR-mediated miniature inhibitory postsynaptic currents (Moscato et al. 2014).
Studies using patient serum or CSF report a strong binding of NMDAR-AB to the hippocampal region, with no evidence of neuronal loss as consequence (Dalmau et al. 2008, Hughes et al. 2010). In the hippocampus, the binding pattern of patient’s NMDAR-AB is dependent on NMDAR density, with higher intensities observed in proximal dendrites of the dentate gyrus (Hughes et al. 2010). In line with the pivotal role of NMDAR signalling in mediating synaptic plasticity in the hippocampus, long term effects in anti-NMDAR
32 INTRODUCTION encephalitis patients include deficits in executive function and memory (Finke et al. 2012, Planaguma et al. 2015).
Presence of NMDAR-AB has been associated with contact with influenza A and B, CNS herpes simplex virus infection, a diagnosis of ovarian teratoma, and a genome-wide significant marker (rs524991) in the proximity of nuclear factor I A (NFIA) gene, a transcription factor that mediates neuroprotection upon NMDAR activation (Dalmau et al. 2007, Zheng et al. 2010, Pruss et al. 2012, Armangue et al. 2014, Hammer et al. 2014, Pruss et al. 2015). Regardless of the triggering events leading to autoantibody production, NMDAR- AB seem to be generated in secondary lymphoid organs and potentially gain access to the CNS upon BBB disruption or via the choroid plexus. This hypothesis is supported by NMDAR-AB seropositivity in healthy individuals (Busse et al. 2014, Dahm et al. 2014). In the lymph nodes, antigen-presenting cells expose naïve B cells to NMDAR that differentiate into memory B cells and antibody-producing plasma cells. Plasma cell-secreted NMDAR-AB and circulating memory B cells can potentially access the brain. NMDAR-AB can exert their effects by direct contact with their target antigens and memory B cells can undergo re- stimulation, antigen-driven affinity maturation, clonal expansion, differentiation into antibody- producing plasma cells and ultimately engage intrathecal production of NMDAR-AB (Moscato et al. 2014, Dalmau 2016).
Post mortem or biopsy histopathological studies of anti-NMDAR encephalitis patients revealed that complement-mediated cell death mechanisms are not related with the pathogenesis of the disease. NMDAR-AB (IgG1) are able to fix complement in vitro, however, no evidence of complement deposition and only residual neuronal and glial cell death has been reported (Martinez-Hernandez et al. 2011, Bien et al. 2012). Overall, there is a low density of inflammatory cells in the parenchyma with the majority of them locating in perivascular and Virchow-Robin spaces. In fact, B and T cell lymphocytic perivascular cuffing along with the presence of antibody secreting plasma cells or plasmablasts (CD138+) and microglial activation are the main histopathological findings reported thus far (Camdessanche et al. 2011, Martinez-Hernandez et al. 2011, Bien et al. 2012). The presence of CD138+ cells in the parenchyma supports additional intrathecal synthesis of NMDAR-AB and the fact that memory B cells can migrate to the brain, cross the BBB and differentiate to antibody secreting plasma cells. Both mouse and human studies have reported immunoglobulin deposition in the brain upon exposure to NMDAR-AB (Martinez-Hernandez et al. 2011, Bien et al. 2012, Planaguma et al. 2015, Wright et al. 2015).
33
INTRODUCTION
Scope of the present work
In the introduction of this thesis, the synergetic interaction between the nervous and immune systems and the potential pathological outcomes mediated by autoimmune processes targeting the brain was addressed, with a particular focus on autoantibodies targeting NMDAR in the context of anti-NMDAR encephalitis.
The first two projects were designed to understand the role of these autoantibodies beyond this pathological condition and gain insight to its effects upon access to the brain. Specifically, Project I aimed at (i) determining the functional properties of NMDAR-ABs of different isotypes; for this purpose a new assay employing human induced pluripotent stem cell-derived neurons was developed. (ii) Identifying which NMDAR epitopes are recognized by these autoantibodies. Project II focused on (i) determining if these NMDAR-AB are present and functional in other mammal species; (ii) assessing the protective role of the BBB and the effects of endogenously produced NMDAR-AB on the brain, in the presence of an open BBB.
Additionally, I have briefly mentioned that disruption of the balance between excitation and inhibition in the brain can contribute to brain diseases as autism and schizophrenia. The contributors for such disruption are not completely understood and might have a common ground between diseases. In Project III, we focused on dissecting the relationship between the severity of autistic traits in schizophrenic patients and imbalances in excitation and inhibition. Specifically, using transcranial magnetic stimulation (TMS), we aimed at determining if individuals with low severity of autistic traits and individuals with high severity of autistic traits would differ in terms of glutamatergic or GABAergic neurotransmission.
35
2. ESTABLISHING A CELL CULTURE SYSTEM FOR TRANSLATIONAL STUDIES
2. ESTABLISHING A CELL CULTURE SYSTEM FOR TRANSLATIONAL STUDIES
To address the aims of the projects aforementioned, with a particular focus on the translational potential of project I and II, a new methodology was implemented. The generation of induced pluripotent stem cell (IPS)-derived neurons from human fibroblasts has been previously described (Shi et al. 2012). To implement this method, different molecular biology techniques were applied in a workflow that comprised reprogramming of fibroblasts to pluripotent stem cells, several steps of quality control of pluripotency, induction of neuronal differentiation and assessment of neuronal maturity and activity (Figure 4). Here, it will be briefly described how this was achieved, and examples of applications of this tool will be addressed in Projects I and II. Resorting to the Göttingen Research Association for Schizophrenia (GRAS) sample collection, fibroblasts were obtained from five different individuals, complying with Helsinki Declaration, and approved by the Ethics Committees of Georg-August-University, Göttingen. All subjects provided written informed consent.
To achieve pluripotency, fibroblasts were reprogrammed using either Sendai virus (SeV) or the STEMCCA excisable polycistronic lentiviral vector to overexpress the four reprogramming factors: the octamer-binding transcription factor 4 (OCT4), the Kruppel-like factor 4 (KLF4), the sex determining region Y-box 2 (SOX2), and the MYC proto-oncogene (c-MYC) (Figure 4A). These two reprogramming strategies differ essentially in their interaction with the host genome. The STEMCCA system requires the integration of the polycistronic vector into the host genome, under the promoter of the elongation factor 1 alpha (EF1α) gene (Sommer et al. 2009, Sommer et al. 2010). In contrast, the SeV-based vector is a non-integrative system, in which the vectors containing the reprogramming factors replicate in the form of negative-sense single stranded RNA in the cytoplasm of infected cells (Fusaki et al. 2009). Different clones, produced using the two reprogramming strategies, were selected and further expanded for pluripotency assessment (Streckfuss-Bomeke et al. 2013). Our strategy included (i) detection of placental alkaline phosphatase expression; (ii) immunofluorescent detection of a panel of markers specific to human embryonic stem cell physiology and fundamental to maintain an undifferentiated state: OCT4, Nanog, SOX2, podocalyxin (Tra1-60), zinc finger CCHC domain-containing protein 1 (LIN28), and stage- specific embryonic antigen-4 (SSEA4); (iii) detection of pluripotency markers at the RNA level and its upregulation from patient fibroblasts to patient IPS using quantitative real time polymerase chain reaction (qPCR). Expression of nuclear (OCT4) and surface (Tra1-60) pluripotency markers was monitored regularly using flow cytometry to assess the pluripotency level overtime.
39 ESTABLISHING A CELL CULTURE SYSTEM FOR TRANSLATIONAL STUDIES
Neuronal conversion and differentiation was based on a dual SMAD inhibition protocol (Figure 4B) (Shi et al. 2012). Here, IPS cells are exposed to a neural induction medium complemented with SB431542 and dorsomorphin molecules. The synergistic action of these neural fate-inducing molecules selectively blocks the TGF-β and the BMP pathways, promoting differentiation towards the neuroectodermal lineage (Chambers et al. 2009, Zhou et al. 2010). Signalling through the nodal/activin branch of the TGF-β pathway induces mesodermal gene expression in ectodermal cells and activation of the BMP pathway leads to the acquisition of epidermal fates. Conversely, inhibition of both Activin/Nodal and BMP signalling, promotes neuroectoderm specification (Munoz-Sanjuan et al. 2002). Successful neural induction results in the formation of a homogeneous neuroepithelial sheet after ten days, with downregulation of pluripotency markers and upregulation of neural stem cell markers such as paired box 6 (PAX6), OTX1/2, Nestin, forkhead box G1 (FOXG1), empty spiracles homeobox 1 (EMX1) and SOX2. Dissociation of the neuroepithelial sheath leads to rearrangement of neural stem cells (SOX2+) in a rosette-like structure, further expanded by application of the mitogen fibroblast growth factor 2 (FGF2). Neuronal progenitors (doublecortin; DCX+) emerge from these structures during the neurogenesis period. A coordinated differentiation of these progenitors is promoted with the application of the small molecule N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), giving rise to neurons (positive for microtubule-associated protein 2; MAP2+). DAPT inhibits γ- secretase, necessary for canonical Notch signalling (Nelson et al. 2007). The Notch signalling pathway is critical for several aspects of neural development: it promotes the survival of neural stem and progenitor cells and newly generated neurons, helps progenitor cells to maintain their undifferentiated state throughout the neurogenic period and promotes the glial fate in multipotent progenitor cells (Mason et al. 2006, Yaron et al. 2006, Nelson et al. 2007). Thus, transient inhibition of Notch signalling using DAPT, leads to delayed G1/S- phase transition committing cells to neurogenesis and a synchronized differentiation of neural progenitors (Nelson et al. 2007, Borghese et al. 2010). Neuronal identity and maturation was confirmed by combining RNA expression and protein markers for DCX, ß- Tubulin III, MAP2, NeuN, GluN1, CAMKII and Synapsin1 (Figure 4C). Functional analysis by calcium imaging coupled with field stimulation revealed both spontaneous and evoked activity (Figure 5).
This in vitro tool enabled us to add a translational aspect to several projects. Specifically, it allowed to assess the expression of erythropoietin receptor in human stem cells (Ott et al. 2015 – see Appendix) and the effects of autoantibodies targeting the NMDAR, using a receptor endocytosis assay based on human-derived IPS-neurons as described in detail in Protects I and II (Castillo-Gomez&Oliveira et al. 2017, Pan&Oliveira et al. 2018, Ehrenreich 2016 – see Appendix).
40 ESTABLISHING A CELL CULTURE SYSTEM FOR TRANSLATIONAL STUDIES
Figure 4. Reprogramming of human fibroblasts and differentiation into neurons. (A) Cellular reprogramming of fibroblasts collected from patient’s gingiva involved cell transduction via Sendai or lentiviral systems to
41 ESTABLISHING A CELL CULTURE SYSTEM FOR TRANSLATIONAL STUDIES
overexpress the four transcription factors: OCT4, KLF4, SOX2 and c-MYC. Successful reprogramming leads to formation of colonies of IPS cells expressing high levels of hPALP, OCT4, Nanog, SOX2, Tra-1-60, LIN28 and SSEA4 markers. Pluripotent colonies can be selected and adapted to feeder-free conditions. (B) Induction of a neuronal phenotype can be achieved by dual SMAD inhibition, upon which cells form a neural stem cell monolayer. Dissociation of the neuroepithelial sheath leads to rearrangement of neural stem cells into a rosette- like structure, further expanded by application of the mitogen FGF2. Neuronal progenitors emerge from these structures during the neurogenesis period and are further differentiated with the application of the small molecule DAPT to give rise to mature neurons. (C) Immunofluorescent stainings of neuronal markers over several days after induction of the neuronal phenotype. N represents the number of days after the beginning of dual SMAD inhibition (Castillo-Gomez&Oliveira et al. 2017). hPALP: human placental alkaline phosphatase; qPCR: real time quantitative polymerase chain reaction; FGF2: fibroblast growth factor 2; DAPT: N-[N-(3,5-Difluorophenacetyl)-L- alanyl]-S-phenylglycine t-butyl ester;
Figure 5. Calcium imaging and field stimulation of N65 IPS-derived neurons labelled with Fluo-4 AM calcium dye. (A) Spontaneous activity was detected with transient changes in fluorescence intensity, over a period of 300 seconds. The number of times the emission of fluorescence surpassed the baseline fluorescence and decayed to close to baseline was determined as a peak of activity. In the bottom figure, the temporal colour code represents the asynchronous nature of the spontaneous activity and some cells with no spontaneous activity
42 ESTABLISHING A CELL CULTURE SYSTEM FOR TRANSLATIONAL STUDIES
(white) for the recorded period. (B) Evoked activity – excitability protocol: cells were stimulated at a constant frequency of 20 Hz with an increasing series of action potentials (APs) triggered from 5 to 40, in a total of four stimuli. The majority of the cells were responsive to the four stimuli (line graph) with stronger responses to the highest number of APs (temporal colour code figure; pink). (C) Evoked activity – stimulation protocol: cells were stimulated with a constant number of APs (20) at an increasing frequency ranging from 5 to 100 Hz, in a total of six stimuli. The majority of the cells were responsive to the six stimuli (line graph) with stronger responses at higher frequencies (temporal colour code figure; pink). The line graphs display the results of one clone from two different individuals and represent the number of peaks recorded during the stimulation period, and its amplitude as readout of the area under the curve (AUC). Temporal colour code is displayed in seconds. Irresponsive cells are labelled in white.
43
3. PROJECT I
3. PROJECT I – All naturally occurring autoantibodies against the NMDA receptor subunit GluN1 have pathogenic potential irrespective of epitope and immunoglobulin class
Overview of Project I
The involvement of NMDAR-ABs in anti-NMDAR encephalitis has been mentioned in the introduction. Despite the pathogenic role attributed to NMDAR-AB in this context, circulating NMDAR-AB, along with other brain-reactive autoantibodies, have been detected in the serum of healthy individuals and with other conditions. These included Parkinson’s and Alzheimer’s disease, schizophrenia, affective disorders, stroke, amyotrophic lateral sclerosis and cancer (Levin et al. 2010, Brimberg et al. 2013, Busse et al. 2014, Dahm et al. 2014, Doss et al. 2014, Steiner et al. 2014, Schou et al. 2016, Finke et al. 2017, Jezequel et al. 2017). Indeed, seropositivity for NMDAR-AB has been reported with an overall seroprevalence of ~10%, where the isotypes IgM (~7%) and IgA (~5%) are more frequently detected than IgG (~1%) (Dahm et al. 2014, Finke et al. 2017). Effects of IgG NMDAR-AB have been assessed in vitro using rat hippocampal neurons and rodent brain sections or in vivo by intraventricular CSF IgG+ infusion in mice (Dalmau et al. 2007, Moscato et al. 2014, Planaguma et al. 2015). In both cases, effects of autoantibody exposure led to reduced surface expression of NMDAR1 synaptic clusters in both inhibitory and excitatory neurons (Moscato et al. 2014, Planaguma et al. 2015). Moreover, in vivo experiments demonstrated acquisition of progressive anhedonic/depressive-like behaviours and memory deficits upon exposure (Planaguma et al. 2015). Further studies confirmed the functional effect of NMDAR-AB of other isotypes in vitro and in vivo (Pruss et al. 2012, Hammer et al. 2014). Given the high seroprevalence of NMDAR-AB in healthy and diseased individuals, it is of outmost relevance to understand their potential pathogenic role. Therefore, this project aimed at a thorough characterization of NMDAR-AB at the functional and molecular level. To do so, a set of NMDAR-AB seropositive and seronegative human samples was selected to cover the immunoglobulin isotypes IgM, IgA and IgG and a spectrum of diseased and healthy individuals. These samples were part of a systematic screening of a large number of individuals for the presence of NMDAR-AB of IgG, IgA and IgM isotypes and determination of their seroprevalence (Dahm et al. 2014, Hammer et al. 2014, Zerche et al. 2015).
Two different approaches were employed to assess NMDAR-AB functionality in vitro: first the ability of all NMDAR-AB isotypes to internalize the NMDAR in human IPS cell-derived neurons was tested and secondly their ability to alter glutamate-evoked responses in Xenopus laevis transfected oocytes. Since all previous studies employed rodent cultures to
47 PROJECT I
assess functionality, a receptor endocytosis assay based on human IPS cells that underwent neuronal differentiation and express the GluN1 subunit of NMDAR was developed (for details see “Establishing a cell culture system for translational studies”). In this live cell-based assay, exposure to all NMDAR1-AB+ dialysed sera led to receptor endocytosis. A significant decrease in cell-surface fluorescence intensity ratio 37°C/4°C was detected as readout of a reduction in surface expression of NMDAR, while negative sera had no effect. This effect was also present when the impact of immunoglobulin isotypes was assessed separately (Castillo-Gomez&Oliveira et al. 2017). To investigate the impact of NMDAR1-AB on receptor activity, glutamate evoked responses were evaluated in Xenopus laevis oocytes co- expressing human GluN1-1/GluN2A subunits using two-electrode voltage clamp. After exposure of oocytes to human dialyzed sera, the area under the curve of the glutamate- evoked current was significantly lowered in seropositive compared with seronegative samples. This effect was sustained for at least 16 minutes in all seropositive samples regardless of their isotype, with no significant differences between isotypes (Castillo- Gomez&Oliveira et al. 2017). With these two experiments it was possible to demonstrate that (i) NMDAR-AB of IgG, IgA and IgM isotypes lead to receptor endocytosis and (ii) consequently reduce the cell response to glutamate.
After identifying the functional effect of NMDAR-AB, the interaction between these autoantibodies and NMDAR were characterized at the molecular level. Based on the concept of seropositivity detection using a cell based assay, HEK293 cells transfected with a series of mutated or chimeric GluN1-1b receptors using the GluN2B as backbone were used. The seven constructs generated allowed to test epitope recognition in the extracellular, transmembrane and intracellular regions of the NMDAR (Castillo-Gomez&Oliveira et al. 2017). Epitope mapping using different GluN1-1b constructs revealed binding to epitopes located in the extracellular ligand-binding domain and NTD, the intracellular CTD and extra- large pore domains (xlp) with no particular disease or isotype-related pattern. Overall, in both monospecific and polyspecific sera involvement of extracellular epitopes was more frequent (11/14). Monospecific and monoclonal sera reactive with intracellular epitopes (3/14) was less frequent and also reactive with GluN2A subunit. The NTD G7 glycosylation site, reported to be important for NMDAR-AB epitope recognition in anti-NMDAR encephalitis, was recognized in 2/10 sera binding to NTD. Targeting specifically the G7 site, seven additional sera from encephalitis patients were tested. All sera had NMDAR-AB of two immunoglobulin isotypes, challenging the previously described exclusivity for IgG associated with this condition and not all of them required the G7 site for binding (Castillo- Gomez&Oliveira et al. 2017). Moreover, autoantibodies targeting the GluN2A subunit were also present in these sera, while no positivity was detected regarding the GluN2B subunit. This result is in line with the postnatal developmental switch in NMDAR conformation, where
48 PROJECT I the GluN2A subunit becomes the preferential partner of the GluN1 subunit upon synaptic maturation leading to a decrease in expression of GluN2B in the adult brain (Gladding et al. 2011, Paoletti et al. 2013).
In this project, NMDAR-AB of three immunoglobulin isotypes (IgM, IgG and IgA) regarding in vitro functionality and epitope recognition has been analysed. Previous studies have focused on the IgG isotype and its role on the pathophysiology of anti-NMDAR encephalitis (Dalmau et al. 2008, Gleichman et al. 2012, Moscato et al. 2014). Thus, in this work the spectrum of NMDAR-AB effects has been broadened to other immunoglobulin isotypes by demonstrating their ability to promote receptor endocytosis and reduce glutamate-evoked responses. The diversity of epitopes recognized by NMDAR-AB demonstrated here, points to a more diverse interaction of these autoantibodies with NMDAR and overcomes the relevance credited exclusively to the G7 site of the NTD.
The presence of these autoantibodies in healthy individuals along with an increase in seroprevalence upon ageing points to the involvement of the BBB in their pathogenicity (Hammer et al. 2014). An intact BBB might pose as the ultimate obstacle for NMDAR-AB to exert their effects in the CNS. In this sense, the consequences of a compromised BBB in the context of NMDAR-AB are the focus of the next project.
Original publication
Castillo-Gómez E*, Oliveira B*, Tapken D*, Bertrand S, Klein-Schmidt C, Pan H, Zafeiriou P, Steiner J, Jurek B, Trippe R, Prüss H, Zimmermann WH, Bertrand D, Ehrenreich H, Hollmann M. All naturally occurring autoantibodies against the NMDA receptor subunit NR1 have pathogenic potential irrespective of epitope and immunoglobulin class. Molecular Psychiatry. 2017; 22(12):1776-1784
*Authors with equal contribution
Personal contribution: As one of the first authors of this manuscript I was involved in establishing the protocol to differentiate human IPS cells into cortical neurons and establishing and performing the NMDAR endocytosis assay to determine the functional effect of human NMDAR autoantibodies in all samples reported in this study. I have also contributed to the analysis and interpretation of the data. Additionally, I was involved in writing the methods and results sections of this paper, helped preparing the figures and figure legends and contributed to the overall preparation of the manuscript. Moreover, I was significantly involved on the study design, experiment planning, literature searches as well as rebuttal and revision.
49
OPEN Molecular Psychiatry (2016) 00, 1–9 www.nature.com/mp
ORIGINAL ARTICLE All naturally occurring autoantibodies against the NMDA receptor subunit NR1 have pathogenic potential irrespective of epitope and immunoglobulin class
E Castillo-Gómez1,9, B Oliveira1,9, D Tapken2,9, S Bertrand3, C Klein-Schmidt2,HPan1, P Zafeiriou4, J Steiner5, B Jurek6,7, R Trippe2, H Prüss6,7, W-H Zimmermann4, D Bertrand3, H Ehrenreich1,8,10 and M Hollmann2,10
Autoantibodies of the IgG class against N-methyl-D-aspartate-receptor subunit NR1 (NMDAR1) were first described in anti-NMDAR encephalitis and seen as disease indicators. Recent work on together over 5000 individuals challenged this exclusive view by showing age-dependently up to 420% NMDAR1-autoantibody seroprevalence with comparable immunoglobulin class and titer distribution across health and disease. The key question therefore is to understand the properties of these autoantibodies, also in healthy carriers, in order to assess secondary complications and possible contributions to neuropsychiatric disease. Here, we believe we provide for human NMDAR1-autoantibodies the first comprehensive analysis of their target epitopes and functionality. We selected sera of representative carriers, healthy or diagnosed with very diverse conditions, that is, schizophrenia, age-related disorders like hypertension and diabetes, or anti-NMDAR encephalitis. We show that all positive sera investigated, regardless of source (ill or healthy donor) and immunoglobulin class, provoked NMDAR1 internalization in human-induced pluripotent stem cell- derived neurons and reduction of glutamate-evoked currents in NR1-1b/NR2A-expressing Xenopus oocytes. They displayed frequently polyclonal/polyspecific epitope recognition in the extracellular or intracellular NMDAR1 domains and some additionally in NR2A. We conclude that all circulating NMDAR1-autoantibodies have pathogenic potential regarding the whole spectrum of neuronal NMDAR-mediated effects upon access to the brain in situations of increased blood–brain–barrier permeability.
Molecular Psychiatry advance online publication, 9 August 2016; doi:10.1038/mp.2016.125
INTRODUCTION chance of displaying NMDAR1-AB seropositivity.11 Disease groups, Circulating autoantibodies (AB) directed against brain epitopes ranging from schizophrenia and major depression, over multiple have long been documented, mainly in connection with classical sclerosis, Parkinson’s and Alzheimer’s disease, to hypertension, autoimmune diseases or paraneoplastic syndromes.1–4 AB target- diabetes and stroke, as well as healthy individuals, share not only ing the N-methyl-D-aspartate-receptor subunit NR1 (NMDAR1; similar NMDAR1-AB seroprevalence but also immunoglobulin (Ig) new nomenclature GluN1 disregarded here for consistency with the class distribution (IgM, IgA and IgG) and titer range.9–11,13 These respective literature, except in the molecular biological section) have unexpected results raised the question of functionality and attracted considerable attention lately. NMDAR1-AB of the IgG relevance of the highly seroprevalent NMDAR1-AB. In translational class were first described in connection with a condition named mouse studies, similar effects of the different classes (IgG, IgM and 5 fl anti-NMDAR encephalitis and induced a ood of publications, IgA) of NMDAR1-AB on behavioral readouts were observed.9 among them many case reports. In several of them, immunosup- 6–8 Likewise, in a human study, an equivalent impact of circulating pressive treatment of seropositive subjects is recommended. NMDAR1-AB of all three isotypes on evolution of lesion size after Anti-NMDAR encephalitis symptoms typically include psychosis, ischemic stroke was noticed.11 Detectable neuropsychiatric cognitive decline, seizures, dyskinesia, decreased consciousness consequences of circulating NMDAR1-AB of all three classes were and autonomic instability.5 These symptoms are reminiscent of restricted to individuals with compromised blood–brain–barrier, those found upon NMDAR antagonism by ketamine, MK801, or for example, ApoE/APOE carrier status, both clinically and related drugs, and have been explained by reduced surface 9,11,15 expression of NMDAR1 upon exposure to NMDAR1-AB.5 experimentally. In studies using rodent hippocampal neu- Rendering the situation more complex, a high age-dependent rons, we found NMDAR1 internalization upon NMDAR1-AB seroprevalence of NMDAR1-AB has been recognized recently.9–14 (IgM, IgA, IgG) binding as explanation of its reduced surface 9,15 According to these findings in meanwhile 45000 subjects, any expression. A comparable finding had previously been 5,16 40-year-old person has an ~ 10%, any 80-year-old person an ~ 20% described only for IgG.
1Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany; 2Department of Biochemistry I – Receptor Biochemistry, Ruhr University, Bochum, Germany; 3HiQscreen, Geneva, Switzerland; 4Institute of Pharmacology and Toxicology, University Medical Center, Göttingen, Germany; 5Department of Psychiatry, University of Magdeburg, Magdeburg, Germany; 6Department of Neurology, Charité, University Medicine, Berlin, Germany; 7German Center for Neurodegenerative Disorders (DZNE), Berlin, Germany and 8DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany. Correspondence: Professor H Ehrenreich, Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Hermann-Rein-Strasse 3, Göttingen 37075, Germany. E-mail: [email protected] 9These authors share first authorship. 10These authors share last authorship. Received 18 March 2016; revised 3 May 2016; accepted 1 June 2016 NMDAR1-autoantibody epitopes and functionality E Castillo-Gómez et al 2 Considering the high seroprevalence of NMDAR1-AB across and reduced glutamate-evoked currents. The AB recognize health and disease, the key question is to understand the epitopes in the extracellular and/or intracellular NMDAR1 domains properties of these AB, also in healthy carriers, in order to assess and, surprisingly, some positive sera also in the NR2A subunit of secondary complications and possible contributions to neuropsy- NMDAR. Thus, most intriguingly, they all have potentially (patho) chiatric disease, for example, cognitive decline, psychotic symp- physiological relevance regarding brain functions. toms or epileptic seizures. Are they like a ‘ticking time bomb’ once the blood barrier opens? MATERIALS AND METHODS Here, we believe we provide for human NMDAR1-AB the first All experiments were performed by researchers unaware of group comprehensive analysis of their target epitopes and of function- ‘ ’ ality. We investigated whether NMDAR1-AB of different Ig classes, assignment ( fully blinded ). derived from sera of subjects with very diverse conditions, would (i) reveal functionality in an internalization assay using human Human samples induced pluripotent stem cell (IPSC)-derived cortical neurons; (ii) Serum specimens (N=29) from our phenotyping/biomarker trials8,10,11,17 lead to electrophysiological consequences in NR1-1b/NR2A- were selected to (i) cover a spectrum of diseases and health, (ii) include all expressing Xenopus oocytes; and (iii) be directed against the NMDAR1-AB Ig classes and (iii) build on enough material for extensive testing (Table 1). In addition, samples (N=7; few μl available) for targeted same or against different epitopes of NMDAR1. epitope mapping were obtained from anti-NMDAR encephalitis patients We report as most important take-home message that, (Charité, HP). Subject data/human materials (including IPSC) were collected independent of any medical condition or Ig class, NMDAR1-AB according to ethical guidelines/Helsinki Declaration including subjects’ are functional, leading to decreased NMDAR surface expression informed consent.
Table 1. Overview of donors of NMDAR1-AB-positive and -negative serum samples
Seropositive individuals (N = 14) Seronegative individuals (N = 15) P-value
Gender, No. (%), womena 10 (71.4) 7 (46.7) 0.176 Age at examination, mean ± s.d., yearsb 62.87 ± 24.44 65.24 ± 10.99 0.234
Diagnosis, No. (%)a 9 Healthy 2 (14.3) 4 (26.7) > > NMDAR1-AB encephalitis 2 (14.3) 0 (0.0) > > Psychiatric conditionsc 1 (7.1) 1 (6.3) = Diabetes 0 (0.0) 2 (13.3) 0.418 > Hypertension 7 (50.0) 6 (40.0) > > Diabetes and hypertension 2 (14.3) 1 (6.7) ;> Other medical conditionsd 0 (0.0) 1 (6.7)
NMDAR1-AB seroprevalence, titers, No.e IgA (1:10; 1:32; 1:100; 1:320; 1:1000; 1:3200) 2; 0; 0; 0; 4; 0 IgG (1:10; 1:32; 1:100; 1:320; 1:1000; 1:3200) 0; 0; 1; 1; 1; 1 n/a IgM (1:10; 1:32; 1:100; 1:320; 1:1000; 1:3200) 0; 0; 1; 1; 5; 1
a b Abbreviations: AB, autoantibodies; IG, immunoglobulin; n/a, not applicable; NMDAR1, N-methyl-D-aspartate-receptor subunit NR1. Chi-square test. Mann– Whitney U-test. c‘Psychiatric conditions’ include two schizophrenia patients. d‘Other medical conditions’ include hypercholesterolemia, asthma bronchiale and glaucoma. eNote that of the total sample N = 4 individuals were seropositive for both IgA and IgM.
Figure 1. NMDAR1 endocytosis in human induced pluripotent stem cell (IPSC)-derived cortical neurons after autoantibodies (AB) exposure. (a) Reprogramming strategy: Human fibroblasts obtained from gingiva biopsies were transduced using a lentiviral system to simultaneously integrate the transcription factors OCT4, KLF4, SOX2 and c-MYC. Transcription factor expression leads to cell reprogramming and colony formation of IPSC, which are differentiated into cortical neurons using a dual SMAD inhibition strategy (selective blocking of TGF-β and BMP pathways). Cells acquire neuronal identity forming a neural stem cell monolayer. Dissociation of the neuroepithelial sheath leads to rearrangement of neural stem cells (SOX2+) in a rosette-like structure, further expanded by application of the mitogen FGF2. Immature neurons (DCX+) emerge from these structures during the neurogenesis period to give rise mainly to MAP2+ cortical excitatory neurons. (b)At day 70 of maturation, neurons are exposed to human sera. NMDAR1-AB binding to NMDAR1 (at 4 °C) leads to rapid endocytosis of the receptor–antibody complex at 37 °C. The remaining cell-surface NMDAR1 is detected by combining mouse anti-NMDAR1-AB (step 1) and secondary donkey anti-mouse Alexa 546-AB (step 2). Neurons are defined as NeuN+ cells. (c) Representative confocal images of neurons exposed to an IgG seropositive sample are shown on the left. Both pictures are Z-projections of three consecutive focal planes 0.5 μm apart, taken at × 100 magnification under confocal laser-scanning microscope. The upper picture depicts a neuron (NeuN+, labeled in green), kept at 4 °C (no endocytosis allowed—control condition) and the lower picture a neuron at 37 °C (endocytosis). For each serum sample, cell membrane NMDAR1 (labeled in red; arrow heads) is quantified. Degree of internalization is expressed as ratio of fluorescence intensity measured at both temperatures. Seropositive samples show reduced fluorescence intensity ratio (37 °C/4 °C) compared with seronegative samples (graph on the left; unpaired Student’s t-test: t16 = 11.16; Po0.001), consistent with a decrease in surface NMDAR1 after exposure to NMDAR1-AB. Right graph: Fluorescence intensity ratio is lower for all seropositive samples (separate analyses shown for samples with only IgG, IgM, IgA or IgM+IgA, in purple, orange, green and yellow columns, respectively) and the positive control (IgG M68-AB). One-way ANOVA: F6,17 = 70.59; Po0.001; multiple comparisons with Bonferroni’s post hoc correction, P-valueo0.05; results given as mean ± s.e.m. BMP, bone morphogenic pathway; c-MYC, avian myelocytomatosis virus oncogene cellular homolog; DAPI, 4',6-diamidino-2-phenylindole; DCX, doublecortin; FGF2, fibroblast growth factor 2; KLF4, Krüppel-like factor 4; MAP2, microtubule-associated protein 2; NMDAR1, N-methyl-D- aspartate-receptor subunit NR1; NeuN, neuronal nuclear antigen; OCT4, octamer-binding transcription factor 4; SOX2, sex determining region Y-homeobox 2; STEMCCA, stem cell cassette; TGF-β, transforming growth factor-β.
Molecular Psychiatry (2016), 1 – 9 NMDAR1-autoantibody epitopes and functionality E Castillo-Gómez et al 3 NMDAR1-AB titer determination Dialysis of serum samples Recombinant immunofluorescence tests (Euroimmun, Lübeck, Germany), Functional studies were conducted with sera following ammonium sulfate clinical standard procedures, were used to detect NMDAR1-AB, based on precipitation of immunoglobulins18 and dialysis (Slide-A-Lyzer Mini- HEK293T cells transfected with NMDAR1,5,6 and secondary AB against human Dialysis-Units, 10 000 MWCO Plus Float, Thermo Fisher Scientific, Rockford, IgG, IgM or IgA. Results were independently assessed by three investigators. IL, USA).
REPROGRAMMING OF HUMAN FIBROBLASTS TO IPSC AND DIFFERENTATION TO CORTICAL NEURONS
STEMCCA lentiviral system Day 25 DCX Day 70 MAP2 → → → OCT4 KLF4 SOX2 c-MYC Sox2 DAPI DAPI
TGF- 75 μm 75 μm Transcription factor induction 〈SB431542〉 Human Reprogramming FGF2 fibroblasts (gingiva) Dual SMAD inhibition
→ → Induced pluripotent BMP Neuroepithelial sheet Rosette formation Cortical neurons stem cells (IPSC) 〈Dorsomorphin〉 Neurogenesis
NMDAR1 ENDOCYTOSIS ASSAY NMDAR1-AB binding (4°C) NMDAR1 endocytosis (37°C) Dialyzed human serum Human NMDAR1-AB Human NMDAR1-AB NMDAR1
NMDAR1 Human IPS-derived cortical neurons (day 70)
Fixation, staining Labeling of the remaining surface Labeling of the remaining surface and confocal microscopy NMDAR1 (step 2; 4°C) NMDAR1 (step 1; 4°C) NMDAR1 NeuN DAPI Donkey anti-mouse Mouse Alexa 546 NMDAR1-AB
5 μm NMDAR1 NMDAR1
EFFECT OF NMDAR1-AB ON NMDAR AVAILABILITY AT THE NEURONAL MEMBRANE
Seropositive patient t16=11.16; p<0.001 F6,17=70.59; p<0.001 NMDAR1 NeuN DAPI
4°C
4 μm 37°C
Molecular Psychiatry (2016), 1 – 9 NMDAR1-autoantibody epitopes and functionality E Castillo-Gómez et al 4 Reprogramming of human fibroblasts and differentiation into maintained at holding potential of − 80 mV and compounds/AB incuba- neurons tions conducted in 96-well-microtiter plates (Thermo Fisher Scientific). Only 4 μ Human fibroblasts from gingiva biopsies were reprogrammed using cells displaying low leak current and responses 0.5 A to a test pulse μ μ STEMCCA system (Merck Millipore, Darmstadt, Germany) for introduction containing 0.3 M glutamate/10 M glycine (Sigma-Aldrich) were retained of OCT4, SOX2, KLF4 and c-MYC.19 Clones were tested for pluripotency for successive testing (Figures 2a and b). Cells were then challenged with markers following standard procedures.19 After reprogramming, IPSC was pre-incubation of 120 s in control medium (OR2 medium) followed by brief adapted to feeder-free culture system (Matrigel matrix, Corning, Wiesba- exposures to 0.3 μM glutamate/10 μM glycine (10 s) every 2 min for 10 min den, Germany) and TeSR™-E8™ medium (Stem Cell Technologies, (Figure 2b, steps 1 and 2). The last 4 min was considered as control Cologne, Germany). Neural induction was based on dual SMAD inhibition response-amplitude to glutamate (Figure 2c, right-side graphs, − 4–0 min). (Figure 1a).20 Upon stabilization, cells were exposed for 16 min to dialyzed serum samples (titer dilution for seropositive and 1:1000 for seronegative samples) or to positive control (M68-AB, 1:1000). NMDAR activity was Endocytosis assay tested by brief exposure to 0.3 μM glutamate/10 μM glycine (10 s) every Human IPSC-derived cortical neurons grown on coverslips, 70 days post 2 min for 16 min in presence of the dialyzed serum (Figure 2b, steps 3 neural induction, were pre-cooled on ice (10 min), and washed 3 × with and 4). To evaluate the cellular response, area-under-the-curve (AUC) for cold HBSS (Hank’s balanced salt solution; Life Technologies, Darmstadt, each glutamate-evoked current was computed and normalized to AUC of Germany). Culture media were kept at 37 or 4 °C. Neurons were incubated control responses (−4–0 min). Data were analyzed using custom-made (30 min) in cold HBSS with 1:100 diluted sera (14 seropositive; 6 software (Matlab-Mathworks, Ismaning, Germany). For each sample, a seronegative), control NMDAR1-AB (M68, mouse IgG, SYSY, Göttingen, mean of ⩾ 3 oocytes was calculated (Figure 2c, left-side graphs). Germany) or HBSS alone (negative control). After three HBSS washes to remove unbound AB, neurons were returned to their media and incubated for 20 min at 37 °C (2 coverslips/sample, to allow endocytosis) or 4 °C Epitope mapping using NMDAR1 constructs (1 coverslip/sample, endocytosis control). After ice-cold HBSS wash, NMDAR1 constructs were generated based on the longest splice variant, coverslips were kept on ice (5 min). Remaining surface NMDAR1 was GluN1-1b (GenBank accession #U08263; Figures 3a and b). All cDNAs exposed to a mouse NMDAR1-AB (N-terminal; Abcam, Cambridge, UK, (including GluN2A: #AF001423; GluN2B: #U11419) were cloned into 1:100), 10 min on ice, followed (after ice-cold HBSS wash) by secondary pcDNA4/TO/myc-HisA (Invitrogen, Carlsbad, CA, USA) such that the donkey anti-mouse IgG (Life Technologies, Alexa-Fluor546, 1:100) for encoded receptor had a myc-His-tag connected to its C-terminus by a 10 min on ice in dark, and finally three ice-cold HBSS washes to remove short peptide linker (SRGPF). Chimeras and mutants for epitope analysis unbound AB. Neurons were fixed with ice-cold 4% paraformaldehyde were constructed by overlap-extension-PCR using chimeric or mutagenic 23,24 (30 min) and washed 3 × (5 min) with 0.1 M phosphate-buffered saline primers. To transiently express glutamate receptor subunits, (PBS). Then, cells were blocked and permeabilized for 1 h at RT (5% normal HEK293T cells were cultured in high-glucose DMEM (Life Technologies). goat, 5% normal horse, 5% fetal calf serum, 0.5% Triton X in PBS), double- On a 35-mm dish, 50 000 cells were plated, grown for 3 days, transfected stained at 4 °C overnight with chicken anti-NeuN-AB (SYSY, 1:500) and with receptor cDNA (3 μg) using Metafectene-Pro (Biontex, Munich, secondary donkey anti-chicken AB (Life Technologies, Alexa Fluor 488, Germany) and, 24 h after transfection, seeded onto poly-D-lysine-coated 1:250) for 1 h at RT (all dark) (Figure 1b). Nuclei were visualized using DAPI coverslips (Sigma-Aldrich). At 2 days after transfection, cells were fixed with − 1 (Sigma-Aldrich, Munich, Germany, 0.01 μgml ). After PBS wash, cover- 5% paraformaldehyde (20 min), washed 5 × with 0.1 M PBS, permeabilized slips were mounted on Super-Frost slides with Mowiol-mounting-media with 0.1% Triton X-100 (5 min), again washed 5 × with PBS, blocked for 1 h (Sigma-Aldrich). Confocal laser-scanning microscopy was used to quantify with 5% normal goat serum (Sigma-Aldrich). After 5 × PBS washes, cells cell-surface NMDAR1 density (×63 glycerol objective; TCS-SP5 Leica- were incubated with serum (1:10–1:200) or monoclonal mouse anti-myc Microsystems, Mannheim, Germany). From each coverslip, Z-series of antibody (transfection efficiency control; self-made, purified from mouse- optical sections (0.5 μm apart) covering the three-dimensional extension of clone-9E10) for 1 h, washed 10 × with PBS, incubated for 1 h with Alexa neurons were acquired (sequential scanning mode, identical acquisition Fluor 488-labeled goat anti-human secondary AB (IgG: Southern Biotech, parameters). For analysis, 50 NeuN+ cells/coverslip were randomly selected Birmingham, AL, USA; IgA: Jackson ImmunoResearch, West Grove, PA, USA; 21 using FIJI-ImageJ software. Soma profile including NMDAR1 surface IgM: Thermo Fisher Scientific) or anti-mouse IgG (controls; Thermo Fisher expression was drawn and fluorescence intensity/cell surface area Scientific), washed 5 × with PBS, incubated with TO-PRO-3 iodide nuclear- (Alexa-Fluor546) automatically measured. Background was subtracted, stain (Molecular Probes, Eugene, OR, USA) (15 min), and washed 5 × with mean intensity for each coverslip determined and fluorescence intensity PBS. Cells were mounted in Fluoromount-G (Southern Biotech) and ratio (37 ºC/4 ºC) calculated. analyzed by confocal microscopy (63 × oil objective, TCS SP2 AOBS, Leica- Microsystems). All results are consensus judgements from at least four Oocyte preparation and injection independent investigators of two different laboratories. Xenopus laevis oocytes were prepared, injected and tested using standard procedures. Briefly, ovaries were harvested from Xenopus females in deep Statistics − 1 anesthesia by hypothermia (4 °C) and MS-222 (Sigma-Aldrich, 150 mg l ). All statistical analyses were performed using SPSS for Windows version Animals were decapitated and pithed obeying animal rights (Geneva, 17.0 (IBM-Deutschland, Munich, Germany). Group differences in categorical Switzerland). A small piece of ovary was isolated for immediate preparation and continuous variables were assessed using Chi-square and Mann– while the remaining part was placed at 4 °C in sterile Barth-solution Whitney U-test. One-way or repeated-measures ANOVA, followed by containing [mM]: NaCl 88, KCl 1, NaHCO3 2.4, HEPES 10, MgSO4.7H2O 0.82, multiple pair-wise comparisons with Bonferroni’s post hoc correction, was Ca(NO3)2.4H2O 0.33, CaCl2.6H2O 0.41, at pH 7.4, supplemented with employed to determine the significance of fluorescence intensity or AUC; − 1 μ − 1 100 U ml penicillin/100 gml streptomycin (Life Technologies). Auto- P-values o0.05 were considered as significant; data in figures are matic injection of mRNAs encoding for human NMDAR (NR1-1/NR2A) was mean ± s.e.m. performed in batches of 95 oocytes using Roboinject22 (Multi Channel Systems, Reutlingen, Germany) (Figure 2a). Optimal expression of NMDAR − 1 was obtained with injection of 15 nl containing 0.2 μg μl of RNAs, ratio RESULTS NR1-1b:NR2A = 1:1; mRNA was prepared using mMessage mMachine SP6 transcription kit for NR1-1b and T7 for NR2A (Thermo Fisher Scientific). In human IPSC-derived cortical neurons, exposure to all NMDAR1- AB-positive sera tested, independent of Ig class and titer, led to receptor endocytosis, reflected by decreased cell-surface Electrophysiological recordings and experimental protocol fluorescence intensity ratio 37 °C/4 °C (perikaryal labeling). Nega- NMDAR activity was validated 60 h after mRNA injection by 2-electrode tive sera had no effect (unpaired Student’s t-test: t16 = 11.16; voltage clamp (Figure 2a). All electrophysiological recordings were o performed using an automated HiClamp system (Multi Channel Systems) P 0.001; seronegative samples: n=6, neurons n=900; seroposi- at 18 °C and with cells superfused with OR2 medium (mM: NaCl 88, KCl 2.5, tive samples: n=14, neurons n=2100) (Figure 1c). Analyzing the fi HEPES 5, CaCl2.2H2O 1.8, pH 7.85). To chelate zinc contaminant, 10 μM impact of Ig classes separately, signi cant results remained for all EDTA was added to all solutions. Unless otherwise indicated, cells were (one-way ANOVA: F6,17 = 70.59; Po0.001 with Bonferroni post hoc
Molecular Psychiatry (2016), 1 – 9 NMDAR1-autoantibody epitopes and functionality E Castillo-Gómez et al 5 a OOCYTE PREPARATION AND VALIDATION b EXPERIMENTAL PROTOCOL hNR1-1b/NR2-A 1 2 mRNAs Holding voltage + I - + Control Holding Glu+Gly - voltage medium + (10s; every 2min 60h V (120s) I - for 10min) + - NR2-A GND V NR1-1b GND Dialyzed Oocyte Glu+Gly+serum NR2-A (10s; every 2min NR1-1b human serum for 16min) (120s)
Test pulse: Glu(0.3μM) + Gly(10μM) Confirmation of 4 3 NMDAR activity
c EFFECT OF NMDAR1-AB ON GLUTAMATE-EVOKED RESPONSE Control medium Dialyzed human serum Time*Group -6 : F7,189=7.43; p<10 0
-2 µA -4
-6 IgG seropositive patient (MAKÜ) -4 0 4 8 12 16 Time (min)
0
-2 µA -4 ECORDINGS
R -6 IgM seropositive patient (JOMA) -4 0 4 8 12 16 Time (min) LAMP
C 0
-2 µA -4 OLTAGE V -6 IgA seropositive patient (GESC) -4 0 4 8 12 16 Time (min)
0 RAPHS OF
G -2 µA -4 AMPLE
S -6 Positive control (M68-AB) -4 0 4 8 12 16 Time (min)
0
-2 µA -4
-6 Seronegative patient (HAPE) -4 0 4 8 12 16 Time (min)
Figure 2. Decreased NMDAR activity after autoantibodies (AB) exposure. (a) NMDAR activity in Xenopus laevis oocytes expressing human NR1-1b/NR2A is confirmed using 2-electrode voltage clamp recordings. (b) Control glutamate response of each oocyte is tested after 120 s incubation in control medium followed by 10 s exposure to glutamate and glycine every 2 min for 10 min (steps 1 and 2). Cells are afterwards exposed for 120 s to dialyzed serum samples or to positive control (M68-AB), followed by 10 s exposures to glutamate and glycine every 2 min for 16 min (steps 3 and 4). (c) An increase in AUC of the glutamate-evoked response starting at 6 min and lasting for at least 16 min is observed in seronegative but not in seropositive samples (left upper graph; one-way repeated-measures ANOVA: time × group interaction: − 6 F7,189 = 7.43, Po10 ; Bonferroni’s post hoc correction for multiple comparison: only P-values o0.05 are shown). The left lower graph shows curves for Ig classes separately which all are below the seronegative curve (one-way repeated-measures ANOVA: time × group interaction: F35,168 = 1.98, P=0.002). On the right side, five representative recordings of cells exposed to IgG, IgM and IgA seropositive samples, positive control sample (M68-AB) and a seronegative sample are shown. The first two curves of every graph show the last 4 min of the control response to glutamate (see Materials and methods for detailed information). AUC, area-under-the-curve; Glu, glutamate; Gly, glycine; GND, signal ground; I, intensity; Ig, immunoglobulin; NMDAR, N-methyl-D-aspartate receptor; V, voltage.
Molecular Psychiatry (2016), 1 – 9 NMDAR1-autoantibody epitopes and functionality E Castillo-Gómez et al 6
a TOPOLOGY & DOMAIN STRUCTURE OF NMDAR1 (GluN1-1b) CONSTRUCTS
b AMINO ACID SEQUENCE & DOMAINS OF GluN1-1b
c NTD/G7 EPITOPES RECOGNIZED BY ANTI-NMDAR ENCEPHALITIS SERA NMDAR1-AB ID(a) AGE(b) GENDER GluN1-1b GluN1-1b-NTD GluN1-1b-G7 IG CLASS TITER EPITOPE IgG 1:100 NTD (G7) + - - HP1 21 F IgM 1:10 Other + + + IgG 1:10 NTD (G7) + - - HP2 18 F IgM 1:100 Other + + + IgG 1:100 NTD + - + HP3 30 F * IgM 1:100 Other + + + IgG 1:100 Other + + + HP4 19 F * IgM 1:10 Other + + + IgG 1:10 NTD (G7) + - - HP5 27 F IgM 1:100 Other + + + IgG 1:100 NTD (G7) + - - HP6 22 F * IgA 1:1000 other + + + IgG 1:100 Other + + + HP7 19 F * IgA 1:10 Other + + + (a) Scrambled identification code; (b) Age at examination (years); *cases with ovarian teratoma. Figure 3. GluN1-1b constructs for epitope mapping of NMDAR1-AB. (a) Topology and domain structure of the GluN1-1b constructs used. The scheme on the left side shows the membrane topology of the GluN1-1b subunit, with domains colored as in (b). Chimeras and mutagenic constructs targeting the different domains of GluN1 are explained on the right (all residue numbers include the signal peptide; GluN1 and GluN2B constructs were generated based on GenBank accession numbers U08263 and NM_012574, respectively). (b) Amino-acid sequence and domains of GluN1-1b. The position marked in the sequence as G7 (glycosylation site 7) is the residue to which the oligosaccharide is attached (N389). The recognition site for this type of glycosylation is N-X-S/T; therefore, mutation of T391 to A in the construct number 2 (GluN1-1b-ΔG7) prevents glycosylation 2 residues upstream of it (N389). (c) NTD/G7 epitopes recognized by serum NMDAR1-AB of different Ig classes from seven female patients with diagnosed anti-NMDAR encephalitis. AB, antibodies; CTD, C-terminal domain; L1, intracellular loop 1; L2, intracellular loop 2; LBD, ligand-binding domain; NMDAR1, N-methyl-D-aspartate-receptor subunit NR1; NTD, N-terminal domain; P, ion channel pore; TMD, transmembrane domains; xlp, extra-large pore domain.
Molecular Psychiatry (2016), 1 – 9 NMDAR1-autoantibody epitopes and functionality E Castillo-Gómez et al 7
Figure 4. NMDAR1-AB recognize several GluN1-1b epitopes. The figure-table, summarizing the results of NMDAR1-AB epitope mapping, includes only seropositive individuals. Upper left: Representative tilescan confocal images of HEK293T cells transfected with NMDAR1 as used for serum testing are shown. NMDAR1-AB seropositivity (left) and seronegativity (right) is illustrated for IgM as example (anti-human IgM FITC-labeled as secondary antibody). Insets are × 4 magnifications of the squared areas in their respective images. All images are Z-projections of 10 consecutive focal planes located 0.5 μm apart and were taken under a confocal laser-scanning microscope using × 100 oil objective (Leica TSC-SP5). Upper right: Schematic drawings of the different constructs used to transfect HEK293T cells (compare Figure 3). The first two constructs represent the complete version of the GluN1-1b and GluN2B subunits of NMDAR. The next seven constructs present deletions, mutations or replacements of defined regions, allowing identification of the epitopes recognized by NMDAR1-AB from healthy and ill individuals. Positivity (+) or negativity (− ) for every construct in every sample is listed underneath. On the very right side, anti-GluN2A seropositivity/Ig class is listed (anti-GluN2A titers range from 1:10 up to 1:200). AB, autoantibodies; CTD, C-terminal domain; Ig, immunoglobulin; NMDAR1, N-methyl-D-aspartate-receptor subunit NR1; NTD, N-terminal domain. test P-values: IgG: Po0.001; IgM: Po0.001; IgA: P=0.012 and IgM GluN2A-AB (9/14) was frequently detected in the NMDAR1-AB- +IgA: Po0.001). positive sera (Figure 4). Separate exploratory analyses of GluN2A- To investigate the impact of NMDAR1-AB on activity, glutamate- AB carrier versus non-carrier sera regarding internalization assay evoked responses were evaluated in Xenopus laevis oocytes co- and electrophysiology results did not reveal differences. Overall, expressing human NR1-1/NR2A subunits. Only 6 min after no particular disease-related pattern appeared. exposure of oocytes to human sera, the AUC of the glutamate- The G7 site of the NTD, an epitope believed to be crucial for evoked response was significantly lower in seropositive compared NMDAR1-AB found in encephalitis,25 was recognized in 2/10 sera with seronegative samples. This effect was sustained for at least binding to NTD (Figure 4, negativity for construct 2). Since these 16 min (Figure 2c; one-way repeated-measures ANOVA: time × sera were from two anti-NMDAR encephalitis cases reported − 6 8 group interaction: F7,189 = 7.431, Po10 ; Bonferroni post hoc previously (without epitope mapping), we tested another seven correction for multiple comparison: only P-values o0.05 are sera from anti-NMDAR encephalitis patients specifically for this shown). Evaluating Ig classes separately, the significant global epitope. All seven sera had NMDAR1-AB of two Ig classes. Epitope effect remained (one-way repeated-measures ANOVA: time × location in NTD was seen in 5/7 sera for IgG, with 4/7 recognizing group interaction: F35,168 = 1.98; P=0.002; Figure 2c, left lower G7. IgM and IgA recognized other epitopes, as did IgG in 2/7 sera graph). (not further determined due to lack of material) (Figure 3c). Epitope mapping using seven different NMDAR1 constructs (Figures 3a and b) revealed recognition by the NMDAR1-AB- positive sera of different epitopes, located in the extracellular DISCUSSION ligand-binding domain and N-terminal domain (NTD) as well as The present paper systematically analyzed for the first time the intracellular C-terminal domain (CTD) and extra-large pore NMDAR1-AB of three Ig classes (IgM, IgG and IgA), derived from domain (xlp). NMDAR1-AB seropositivity was polyclonal/polyspe- randomly selected individuals of different age, gender and cific in 7/14 sera and likely mono- or oligoclonal/oligospecific medical condition, regarding in vitro functionality and epitope (mainly IgG) in 7/14. Whereas no GluN2B-AB (0/14) was found, location. All NMDAR1-AB-positive sera tested led to NMDAR1
Molecular Psychiatry (2016), 1 – 9 NMDAR1-autoantibody epitopes and functionality E Castillo-Gómez et al 8 internalization in IPSC-derived human cortical neurons and to symptomatic consequences.2 Moreover, inflammation in the brain reduced glutamate-evoked response in NR1-1b/NR2A-expressing likely has a crucial role in determining syndrome acuteness oocytes. Several different epitopes were identified, located in the and severity as contributed by circulating NMDAR1-AB and extracellular part of NMDAR1 (NTD, ligand-binding domain) or respective plasma cells, including boost in AB titers (upon epitope intracellular, CTD and in the xlp, which were recognized by recognition) and class-switch to IgG.33 Especially in individuals NMDAR1-AB of the IgG, IgA and IgM class. Importantly, there was where an overt encephalitis diagnosis is unlikely, determination no consistent functional or epitope pattern detectable regarding of blood–brain–barrier disruption, for example, by a novel Ig class or health/disease state. magnetic resonance imaging method,35 may prove helpful for In light of the comparable functionality of all NMDAR1-AB estimating necessity and benefit of immunosuppressive therapeu- tested here, the high seroprevalence (up to 420%) of NMDAR1-AB tic interventions. is even more puzzling and may indicate a previously unknown dimension of ‘physiological autoimmunity’ that increases with age.9–13 But what are the inducers of such abundant formation of CONFLICT OF INTEREST NMDAR1-AB that potentially influence brain function? So far, The authors declare no conflict of interest. associations were found with certain forms of cancer, mainly ovarian teratoma,5 influenza A and B,9–13 as well as with a genome-wide significant marker on chromosome 1, rs524991,9 ACKNOWLEDGMENTS close to NFIA, a transcription factor mediating neuroprotective This work was supported by the Max Planck Society, the Deutsche Forschungsge- effects of NMDAR.26 There are certainly more hitherto unknown meinschaft (Center for Nanoscale Microscopy & Molecular Physiology of the Brain), predisposing factors for carrying these AB. Broader significance of EXTRABRAIN EU-FP7 and the Niedersachsen-Research Network on Neuroinfectiology NMDAR1-AB is underscored by their presence in mammalians (N-RENNT). The assistance by the UMG-Stem Cell Unit (S. Chen and L. Cyganek) is other than humans.27 This is less surprising in view of the greatly appreciated. substantial seroprevalence also of other brain-directed AB in 28 species like rabbits, pigs and cows. AUTHOR CONTRIBUTIONS While irrespective of the epitope, all 14 NMDAR1-AB-positive sera investigated here (IgM, IgG, IgA) revealed similar AB HE, MH and DB contributed to concept and design of the study. ECG, BO, DT, functionality (internalization, electrophysiology), the AB-inducing SB, RT, PZ, JS, CK, HoP, BJ, HP, WHZ, DB, MH and HE contributed to data factors may have determined the epitopes via mechanisms like acquisition/analysis/interpretation. HE, ECG, BO, DT and MH contributed to molecular mimicry.29,30 Published work on NMDAR1-AB epitopes drafting manuscript and figures. All authors read and approved the final version is scarce25,31,32 and focused on IgG recognizing NTD and the NTD- of the manuscript. G7 domain (N368/G369), probably because this region and Ig class fi 5,25 was rst deemed pathognomonic for anti-NMDAR-encephalitis. REFERENCES Indeed, the sera investigated here of two young females with diagnosed anti-NMDAR-encephalitis, originally reported within a 1 Sutton I, Winer JB. The immunopathogenesis of paraneoplastic neurological 102 – series of schizophrenia cases,8 also recognized this epitope, syndromes. Clin Sci 2002; :475 486. 2 Diamond B, Huerta P, Mina-Osorio P, Kowal C, Volpe B. Losing your nerves? Maybe however, without sticking out functionally (that is, regarding it’s the antibodies. Nat Rev Immunol 2009; 9:449–456. internalization or electrophysiology). Therefore, we analyzed sera 3 Coutinho E, Harrison P, Vincent A. Do neuronal autoantibodies cause psychosis? A of seven additional young female patients, diagnosed with anti- neuroimmunological perspective. Biol Psychiatry 2014; 75: 269–275. NMDAR encephalitis (4/7 with ovarian teratoma), in an NTD-G7- 4 Crisp SJ, Kullmann DM, Vincent A. Autoimmune synaptopathies. Nat Rev Neurosci targeted epitope screen. Interestingly, all 7 subjects carried 2016; 17:103–117. NMDAR1-AB of 2 Ig classes, but only IgG recognized NTD or 5 Dalmau J, Gleichman AJ, Hughes EG, Rossi JE, Peng X, Lai M et al. Anti-NMDA- NTD-G7 in 5/7 sera. Similarly, in a recent study on young females receptor encephalitis: case series and analysis of the effects of antibodies. Lancet with neuropsychiatric manifestation of systemic lupus erythema- Neurol 2008; 7: 1091–1098. tosus, two epitopes in the NTD (outside G7) were recognized by 6 Wandinger KP, Saschenbrecker S, Stoecker W, Dalmau J. Anti-NMDA-receptor encephalitis: a severe, multistage, treatable disorder presenting with psychosis. NMDAR1-AB (only IgG tested). Regrettably, NTD was the only J Neuroimmunol 2011; 231:86–91. mapped region, functionality was not evaluated, and instead of a 32 7 Zandi MS, Irani SR, Lang B, Waters P, Jones PB, McKenna P et al. Disease-relevant cell-based assay, the accepted gold standard, an ELISA was used. autoantibodies in first episode schizophrenia. J Neurol 2011; 258:686–688. Together, these data suggest that factors predisposing young 8 Steiner J, Walter M, Glanz W, Sarnyai Z, Bernstein HG, Vielhaber S et al. Increased women to neuropsychiatric manifestations of NMDAR-associated prevalence of diverse N-methyl-D-aspartate glutamate receptor antibodies in autoimmunity are connected with NTD or NTD-G7 epitopes. The patients with an initial diagnosis of schizophrenia: specific relevance of IgG NR1a accentuated role of IgG in this context is still a matter of antibodies for distinction from N-methyl-D-aspartate glutamate receptor ence- speculation but may be related to inflammation-induced class- phalitis. JAMA Psychiatry 2013; 70: 271–278. switch in the brain.33 Regarding NMDAR1-AB of the IgA class, a 9 Hammer C, Stepniak B, Schneider A, Papiol S, Tantra M, Begemann M et al. single study, mapping epitopes of two female patients, found in Neuropsychiatric disease relevance of circulating anti-NMDA receptor auto- antibodies depends on blood–brain–barrier integrity. Mol Psychiatry 2013; 19: one of them evidence of NTD/G7 as a target epitope (likely among – 31 fi 1143 1149. other epitopes). Interestingly, the signi cance of AB for brain 10 Dahm L, Ott C, Steiner J, Stepniak B, Teegen B, Saschenbrecker S et al. Ser- manifestation of lupus erythematosus seems still debatable. In a oprevalence of autoantibodies against brain antigens in health and disease. Ann recent study, lack of B cells and autoantibodies in a murine model Neurol 2014; 76:82–94. of systemic lupus did not prevent the development of key features 11 Zerche M, Weissenborn K, Ott C, Dere E, Asif AR, Worthmann H et al. Preexisting of neuropsychiatric lupus.34 serum autoantibodies against the NMDAR subunit NR1 modulate evolution of To conclude, all naturally occurring serum NMDAR1-AB lesion size in acute ischemic stroke. Stroke 2015; 46: 1180–1186. obviously have pathogenic potential. For still unexplored reasons, 12 Steiner J, Teegen B, Schiltz K, Bernstein H-G, Stoecker W, Bogerts B. Prevalence of they are highly frequent and their prevalence increases with age. N-methyl-D-aspartate receptor autoantibodies in the peripheral blood: healthy fi control samples revisited. JAMA Psychiatry 2014; 71: 839. NMDAR-AB seropositivity alone de nitely does not justify 13 Castillo-Gomez E, Kastner A, Steiner J, Schneider A, Hettling B, Poggi G et al. The immunosuppressive treatment. Syndromal relevance of serum brain as 'immunoprecipitator' of serum autoantibodies against NMDAR1. Ann NMDAR1-AB depends on accessibility to the brain, that is, Neurol 2015; 79: 144–151. 9,11,13,15 blood–brain–barrier permeability. This permeability might 14 Busse S, Busse M, Brix B, Probst C, Genz A, Bogerts B et al. Seroprevalence of differ regionally, thereby explaining individually variable N-methyl-D-aspartate glutamate receptor (NMDA-R) autoantibodies in aging
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4. PROJECT II
4. PROJECT II – Uncoupling the widespread occurrence of anti-NMDAR1 autoantibodies from neuropsychiatric disease in a novel autoimmune model
Overview of Project II
The crosstalk between the brain and the immune system is a dynamic process where molecules of both systems can influence each other. Although the synergy between both systems is undeniable, when it comes to autoimmune events involving the brain several questions remain unanswered. Presence of serum autoantibodies does not necessarily correlates with CNS disease, and pathogenicity relies on direct contact between the brain antigen and its autoantibody (Nagele et al. 2013). The BBB limits interactions between the CNS and the systemic immune system. During gestation, maternal IgG can cross the placenta, reach the fetal bloodstream and gain access to the brain parenchyma. In mouse, the BBB is permeable to IgG until E17.5 and a similar period of vulnerability to IgG is presumed for the developing human brain (Simister 2003, Braniste et al. 2014). After this developmental period, the BBB is responsible for an almost immunoglobulin free brain parenchyma (Brimberg et al. 2015). In the adult brain, the access of immunoglobulins is restricted to less than 1% of the circulating levels, with the expected transfer over an intact BBB of IgG being 1/500, of IgA 1/600, and of IgM 1/3000 of the serum concentration (Hammer et al. 2014, Crisp et al. 2016).
To circumvent the protective effect of the BBB, the majority of rodent studies addressing the effects of NMDAR-AB in vivo employed brain injection of anti-NMDAR encephalitis patient- derived IgG or CSF in Wistar rats or infusion into the ventricular system of mice (Manto et al. 2010, 2011, Planaguma et al. 2015, Wright et al. 2015). Systemic injection of immunoglobulins (IgA, IgM and IgG) extracted from seropositive individuals in ApoE-/-, presenting a compromised BBB, has also been performed (Hammer et al. 2014). Altogether, these studies highlighted the in vivo functional outcomes of exposure to NMDAR-AB beyond receptor internalization. Increased levels of extra-synaptic glutamate, excitability of the motor cortex and higher seizure propensity to the pro-convulsant pentylenetetrazol emerged as direct consequences of exposure (Manto et al. 2011, Wright et al. 2015). Long-term effects included memory deficits and depressive-like behaviour (Planaguma et al. 2015). In vivo studies extending their focus beyond the IgG isotype, reported a hypersensitive response to MK-801 (Dizocilpine), a GluN1 antagonist, upon systemic application of NMDAR-AB in ApoE- /- (Hammer et al. 2014). Additional experiments using ApoE-/- mice also demonstrated that circulating NMDAR-AB upon accessing the brain parenchyma bind to prefrontal cortex, hippocampus, cerebellum, brain stem and spinal cord and are not detected in the CSF
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(Castillo-Gomez et al. 2016). Interestingly, several brain areas of wild type mice, with an intact BBB, were also positive for NMDAR-AB but at lower titres demonstrating that NMDAR- AB can cross the BBB, possibly by one of the mechanisms mentioned in Figure 2 of the introduction (Castillo-Gomez et al. 2016).
Determination of NMDAR-AB seroprevalence using large cohorts of healthy and diseased individuals pinpointed an increase in seroprevalence upon ageing in humans (Hammer et al. 2014). The presence of natural autoantibodies has been previously described in other species (Marchalonis et al. 1993, Avrameas et al. 2007, DeMarshall et al. 2015, Pruss et al. 2015). To further understand the role of NMDAR-AB, beyond pathogenicity, a systematic screening to determine for the first time its prevalence and age-dependence in dogs, rats, mice, cats, rhesus macaques and baboons has been conducted. Due to the lack of reliable secondary antibodies for some of the species tested, direct immunoglobulin labelling using fluorescein isothiocyanate (FITC) labelled Protein A (Protein A-FITC) was employed to screen cat, dog, rat and monkey sera (Pruss et al. 2015). Protein A is a bacterial Fc receptor present in Staphylococcus aureus and known to bind the Fc-portion of immunoglobulins of different species (Richman et al. 1982, Boyle et al. 1987). Mouse and human samples were screened using IgM, IgA and IgG species-specific secondary antibodies. This assay was cross validated using human serum samples in a two-step procedure: (1) screening with a commercial cell-based assay comprised of GluN1-tranfected HEK293 cells and anti-human isotype-specific secondary antibodies (Dalmau et al. 2008) and (2) immunoglobulin labelling with Protein A-FITC followed by screening with GluN1-tranfected HEK293 cells. Additionally, serum of both monkey species were tested in an independent lab using IgM species-specific secondary antibodies resulting in a 97% concordance of the results obtained in both laboratories. With this screening strategy seropositivity for NMDAR-AB in cats, dogs, rats, mice, rhesus macaques and baboons was detected, extending the list of species with reported NMDAR-AB seropositivity. Moreover, as described in humans, an age-dependent increase in seroprevalence for all species except for monkeys was found. Non-human primates kept in captivity are exposed to chronic life stress (Jacobson et al. 2016) and changes in serum immunoglobulins, particularly IgA, have been reported in individuals with high-stress perception when exposed to psychological stress (Maes et al. 1997). Hence, this inter-species difference might be related with an immunological response to captivity-related stress that leads to the presence of NMDAR-AB since young age in monkeys. Furthermore, in humans that underwent migration, a known stress factor, the previously reported age- dependent increase in seroprevalence is lost. Migrants show an increased seroprevalence of NMDAR-AB at young age when compared to non-migrant individuals of the same age, with IgA being the most frequent isotype.
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Due to the high amino acid sequence homology between the human GluN1 and the species included in this study, the endocytosis assay previously established (see Project I), was applied to test the functionality of these NMDAR-AB. In line with the results obtained for human seropositive samples (Castillo-Gomez&Oliveira et al. 2017), exposure to NMDAR-AB+ sera from all species mentioned above promoted reduction of surface NMDAR demonstrating their functionality.
In anti-NMDAR encephalitis, the presence of NMDAR-AB and an inflammatory brain context mediate the pathophysiological events associated with the disease (Dalmau et al. 2008). Although some triggering events of NMDAR-AB production have been described, it is not clear if the production of these autoantibodies has a pro-inflammatory effect in the brain or if the encephalitic phenotype observed is the result of an additional pathological event (Ehrenreich 2017). Hence, a direct link between NMDAR-AB and inflammation in the brain is missing. The presence of NMDAR-AB in healthy individuals has been previously reported, pointing to the importance of the BBB in preventing them from exerting their pathological effect (Hammer et al. 2014). To clarify the role of the BBB in this context ApoE-/- mice and wild type (ApoE+/+) littermates were immunized with a mixture of four peptide sequences of the extracellular part of GluN1, which included the glycosylation site G7. ApoE-/- mice have been previously described as having an open BBB and blood nerve barrier presenting extensive IgG extravasation in sciatic nerve, cerebellum, spinal cord and cortical and subcortical areas as the hippocampus (Fullerton et al. 2001). Here, the BBB integrity of ApoE-/- mice was reassessed using two fluorescent tracers with different molecular weights: Evans blue (EB: 960.81) and sodium fluorescein (NaFl: 376.28). Increased extravasation of both tracers was detected in ApoE-/- confirming the leakiness of the BBB in these mice. Increased permeability of the BBB does not seem to have an impact in anxiety levels, activity, exploratory behaviour, motor and sensory function, sensorimotor gating, pheromone- based social preference and cognitive performance as determined in a comprehensive behavioural assessment of ApoE-/- and ApoE+/+.
Reduced spontaneous activity in the open field and hyperlocomotion following application of the GluN1 antagonist MK-801 in ApoE-/- mice injected with human serum immunoglobulin extracts of seropositive patients has been previously reported (Hammer et al. 2014). Aiming at assessing the effects of NMDAR-AB in a more physiological context ApoE-/- and ApoE+/+ mice were immunized using a mixture of GluN1 peptides, ovalbumin and Freund’s Adjuvant or ovalbumin and Freund’s Adjuvant as immunization control. In a primary immune response naïve B cells and T cells are mobilized and antibody production peaks in 7 to 10 days upon contact with the antigen. Further B cell activation due to contact with the antigen stimulates naïve or memory B cells to proliferate and differentiate to antibody secreting plasma cells. Upon active immunization, the first antibody isotype produced by a developing B cell is IgM.
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Mature B cells eventually go through class switch recombination events and produce IgA and IgG antibodies (Alberts et al. 2002). Endogenous NMDAR1-AB production was monitored overtime using an ELISA essay. Serum anti-ovalbumin and anti-GluN1 IgG antibodies were first detected between day 5 and 10 after immunization and were present until day 28. The lag phase of antibody production for ovalbumin and GluN1 antibodies was comparable between genotypes pointing to a primary immune response in both groups. This makes the presence of pre-existing memory B cells and a secondary immune response to ovalbumin and GluN1 antigens unlikely and corroborates the negative results for NMDAR-AB obtained with the serum tested prior immunization (pre-immune serum) (Pan&Oliveira et al. 2018).
Immunized and immunization control mice of both genotypes were tested in the open field upon administration of MK-801, 27 days post immunization. MK-801 is a selective non- competitive antagonist of NMDAR known to induce hyperlocomotion (Irifune et al. 1995). In mice, systemic application leads to reduction of NMDAR-mediated neurotransmission and increased extracellular glutamate levels in limbic brain regions. Enhanced non-NMDAR glutamatergic signalling in cortico-striatal circuitry in response to increased glutamate levels is one of the currently accepted explanations for this phenotype (Chartoff et al. 2005). While no changes were detected in baseline locomotion (pre- and post-immunization) between genotype or immunization groups, administration of MK-801 in GluN1 immunized ApoE-/- lead to hyperactive behaviour in the open field. This effect was not found in ApoE-/- immunized with ovalbumin or in ApoE+/+ of both immunization groups. Altogether, these results are in line with the effects of NMDAR-AB observed upon systemic injection in ApoE-/- and demonstrate that the behavioural effects of NMDAR-AB require diffusion of these antibodies to the brain parenchyma through a compromised BBB (Hammer et al. 2014). Thus, the increased hyperactive behaviour revealed in immunized ApoE-/- is a result of the combinatorial effect of NMDAR antagonism by MK-801 and lower NMDAR surface expression due to endocytosis by NMDAR-AB, only possible in the presence of a compromised BBB (Pan&Oliveira et al. 2018).
An inflammatory milieu per se can be detrimental for brain function. To address a potential contribution of NMDAR-AB and an open BBB to the development of an inflammatory reaction in the brain and untangle NMDAR-AB’s contribution to the phenotype, immunohistochemical analysis was performed for both genotype and immunization groups. This analysis focused on the hippocampus as, despite the widespread expression of GluN1 in the CNS, a preferential binding of NMDAR-AB to hippocampal regions has been reported (Dalmau et al. 2008, Hughes et al. 2010). Therefore, the number of Iba1+, CD68+, MHC-II+, CD3+ cells was quantified and a densitometric analysis of GFAP+ was performed in the hippocampus. No significant differences between groups was observed for all markers assessed, pointing to no effect of the immunization with NMDAR-AB or presence of a leaky BBB in microglial cell
66 PROJECT II numbers and activation status as well as T cells or development of astrogliosis. Additionally, no difference in brain’s water content between genotypes was observed, supporting the absence of inflammation in the presence of an open BBB. Altogether, this data suggests that the presence of these NMDAR1-AB alone does not trigger an inflammatory reaction and additional pro-inflammatory events are required to develop an encephalitic phenotype.
To conclude, several mammal species were systematically screened for NMDAR-AB seropositivity and seroprevalence, in young and old age, and for NMDAR-AB functionality. The results of this project broadened the number of species with reported seropositivity for NMDAR-AB and confirmed an age-dependent increase in seroprevalence in cats, dogs, rats and mice not present in monkeys or humans that underwent migration. Furthermore, by establishing a new autoimmune mouse model, with endogenous production of NMDAR-AB, the importance of an intact BBB in preventing NMDAR-AB of exerting its effects in the brain was confirmed and the development of an inflammatory reaction was uncoupled from the effects of NMDAR-AB in brain.
Original publication
Pan H*, Oliveira B*, Saher G*, Dere E, Tapken D, Mitjans M, Seidel J, Wesolowski J, Wakhloo D, Klein-Schmidt C, Ronnenberg A, Schwabe K, Trippe R, Mätz-Rensing K, Berghoff S, Al-Krinawe Y, Martens H, Begemann M, Stöcker W, Kaup FJ, Mischke R, Boretius B, Nave KA, Krauss JK, Hollmann M, Lühder F, Ehrenreich H. Uncoupling the widespread occurrence of anti-NMDAR1 autoantibodies from neuropsychiatric disease in a novel autoimmune model. Molecular Psychiatry. 2018, in press.
*Authors with equal contribution
Personal contribution: As one of the first authors of this manuscript, I was involved in data acquisition and analysis of the experiments supporting the functionally of NMDAR autoantibodies in several mammalian species, including the preparation of human IPS cells derived neurons used to test functionality in vitro. I have analysed the open field data regarding the immunized mice injected with MK-801 and done the immunohistochemistry experiments to assess inflammation in the brain. I was responsible for all the experiments requested for the paper’s revision, which included quantification of CD68+ and MHC-II+ cells in the hippocampus and densitometric analysis of GFAP expression in the CA1 and CA3 hippocampal areas (unpublished data). Additionally, I contributed to the study design and to the overall writing of the manuscript. I wrote the methods section of this paper, prepared the figures and figure legends. I contributed significantly to the revision and the rebuttal letter
67
Molecular Psychiatry https://doi.org/10.1038/s41380-017-0011-3
ARTICLE
Uncoupling the widespread occurrence of anti-NMDAR1 autoantibodies from neuropsychiatric disease in a novel autoimmune model
1 1 2 1 3 1 1 Hong Pan ● Bárbara Oliveira ● Gesine Saher ● Ekrem Dere ● Daniel Tapken ● Marina Mitjans ● Jan Seidel ● 1 1 3 1 4 Janina Wesolowski ● Debia Wakhloo ● Christina Klein-Schmidt ● Anja Ronnenberg ● Kerstin Schwabe ● 3 5 2 4 6 1 Ralf Trippe ● Kerstin Mätz-Rensing ● Stefan Berghoff ● Yazeed Al-Krinawe ● Henrik Martens ● Martin Begemann ● 7 5 8 9 2,10 Winfried Stöcker ● Franz-Josef Kaup ● Reinhard Mischke ● Susann Boretius ● Klaus-Armin Nave ● 4 3 11 1,10 Joachim K. Krauss ● Michael Hollmann ● Fred Lühder ● Hannelore Ehrenreich
Received: 29 August 2017 / Revised: 20 October 2017 / Accepted: 30 October 2017 © The Author(s) 2018. This article is published with open access
Abstract Autoantibodies of the IgG class against N-methyl-D-aspartate-receptor subunit-NR1 (NMDAR1-AB) were considered pathognomonic for anti-NMDAR encephalitis. This view has been challenged by the age-dependent seroprevalence (up to 1234567890 >20%) of functional NMDAR1-AB of all immunoglobulin classes found in >5000 individuals, healthy or affected by different diseases. These findings question a merely encephalitogenic role of NMDAR1-AB. Here, we show that NMDAR1- AB belong to the normal autoimmune repertoire of dogs, cats, rats, mice, baboons, and rhesus macaques, and are functional in the NMDAR1 internalization assay based on human IPSC-derived cortical neurons. The age dependence of seroprevalence is lost in nonhuman primates in captivity and in human migrants, raising the intriguing possibility that chronic life stress may be related to NMDAR1-AB formation, predominantly of the IgA class. Active immunization of ApoE−/− and ApoE+/+ mice against four peptides of the extracellular NMDAR1 domain or ovalbumin (control) leads to high circulating levels of specific AB. After 4 weeks, the endogenously formed NMDAR1-AB (IgG) induce psychosis-like symptoms upon MK-801 challenge in ApoE−/− mice, characterized by an open blood–brain barrier, but not in their ApoE+/+ littermates, which are indistinguishable from ovalbumin controls. Importantly, NMDAR1-AB do not induce any sign of inflammation in the brain. Immunohistochemical staining for microglial activation markers and T lymphocytes in the hippocampus yields comparable results in ApoE−/− and ApoE+/+ mice, irrespective of immunization against NMDAR1 or ovalbumin. These data suggest that NMDAR1-AB of the IgG class shape behavioral phenotypes upon access to the brain but do not cause brain inflammation on their own.
Introduction of these AB, as well as a variably favorable response to immunosuppressive therapy. The reported syndrome, Autoantibodies (AB) of the immunoglobulin G (IgG) class reflecting typical NMDAR1 antagonistic actions, consisted of against the N-methyl-D-aspartate-receptor subunit-NR1 psychosis, epileptic seizures, dyskinesia, cognitive decline, (NMDAR1) were originally interpreted as pathognomonic reduced consciousness, and autonomic dysregulation [1–4]. for a condition called “anti-NMDAR encephalitis”,char- However, work on >5000 individuals, healthy or affected by acterizedbyhighserumandcerebrospinalfluid (CSF) titers different diseases, consistently revealed overall comparable age-dependent seroprevalence of functional NMDAR1-AB of all Ig classes, nurturing serious doubts regarding a purely pathological role of NMDAR1-AB of any Ig class [5–10]. Hong Pan, Bárbara Oliveira, and Gesine Saher contributed equally to NMDAR1-AB apparently belong to a pre-existing this work. autoimmune repertoire [11–17], where Ig isotypes are * Hannelore Ehrenreich determined by extracellular vs. intracellular antigen location [email protected] [6]. This may explain the rarity of the IgG class among AB Extended author information available on the last page of the article directed against extracellular epitopes, e.g., NMDAR1, H. Pan et al.
MOG, and CASPR2. In contrast, AB that recognize intra- men), diagnosed with schizophrenia or schizoaffective disorder cellular antigens, e.g., amphiphysin, ARHGAP26, or according to DSM-IV-TR [24]. Subjects of “extended GRAS” GAD65, show predominance of IgG [6]. Despite this (N = 4933; age 43.29 ± 0.24 years; 56.9% men) comprise apparent “physiological autoimmunity”, no report exists that healthy individuals and patients with different neuropsychiatric systematically screened mammals other than humans for the diagnoses, including schizophrenia, affective disorders, multi- presence of NMDAR1-AB. In recent work, we found that ple sclerosis, Parkinson, ALS, stroke, and personality disorders all naturally occurring NMDAR1-AB are functional and (detailed description in [5, 6, 8, 9]). For this study, subjects are thus have pathogenic potential irrespective of epitope and Ig dichotomously classified as nonmigrants or migrants compris- class [10]. Pathophysiological significance may emerge in ing first (patient migrated) and second generation (parents conditions of compromised blood–brain barrier (BBB), for migrated). Identified migrants (N = 301/N = 4933) are from instance, upon injury, infection, inflammation, or genetic Europe (49.8%), Asia (36.9%), Africa (9%), North America predisposition (APOE4 haplotype), which then allows (2%), South America (0.7%), or mixed (1.6%). substantial access of circulating NMDAR1-AB to the brain where they act as NMDAR antagonists [5, 9, 18–20]. Neurosurgical patients Alternatively, AB-specific plasma cells may reside or settle in the brain and produce large amounts of AB intrathecally A total of N = 72 paired samples of serum and ventricular CSF [14, 21]. The question whether abundant endogenously were available from patients (N = 45 women; age 55.9 ± 2.2 produced NMDAR1-AB of the IgG class can—upon access years; N = 27 men; age 60.2 ± 2.7 years) undergoing neuro- to the brain—induce inflammation and thus “anti-NMDAR1 surgery for various reasons: meningiomas, metastases, and encephalitis” has never been experimentally addressed. other brain tumors (N = 25); intracerebral/subarachnoid The present paper has therefore been designed to (i) sys- hemorrhages (N = 20); hydrocephalus (N = 12); arterial tematically screen mammals other than humans for ser- aneurysms (N = 7); trigeminal neuralgia (N = 4); and others oprevalence of functional NMDAR1-AB and (ii) study mice (N = 4). Most pairs were taken simultaneously at the time point with open BBB behavioral and morphological consequences of surgery, i.e., <5min(N = 64) or <30 min (N = 8) apart. of high circulating levels of endogenous NMDAR1-AB of the IgG class formed in response to immunization. Other mammals
Dogs and cats Materials and methods Serum samples from dogs and cats of different breeds were Ethical approvals prospectively collected during routine (health check/vacci- nation) or diagnostic (spectrum of different disorders) Ethics committees of Georg-August University, Göttingen, workup of outpatients in the Small Animal Clinic, Uni- and collaborating centers approved the Göttingen Research versity of Veterinary Medicine, Hannover. Association for Schizophrenia (GRAS) data collection and other studies “extended GRAS” acquiring human data, Monkeys serum samples, and IPSC [5, 6, 8, 9, 22, 23]. Hannover Medical School Ethics Committee approved the neuro- Serum samples from healthy baboons and rhesus macaques surgical specimen collection. Studies comply with Helsinki were obtained through routine checkups at the Leibniz Declaration. Patients gave written informed consent. Mouse Institute for Primate Research, Göttingen. studies were approved by Animal Ethics (LAVES, Oldenburg) following German Animal Protection Law. Rodents Notes: All experiments were performed by researchers unaware of group assignment. The new nomenclature Serum samples from healthy rats and mice were obtained at GluN1 for NMDAR1 is mostly disregarded here for con- the Max Planck Institute of Experimental Medicine and the sistency with the respective literature. Institute for Multiple Sclerosis Research, Göttingen.
Human samples Serological analyses
GRAS and “extended GRAS” NMDAR1-AB determination by clinical standard procedures
The GRAS [22, 23] subsample used here consists of deep- Human serum and ventricular CSF were tested for phenotyped patients (N = 970; age 39.29 ± 0.40 years; 66.3% NMDAR1-AB positivity using commercially available kits, Anti-NMDAR1 autoantibodies across mammals based on HEK293T cells transfected with NMDAR1 and Endocytosis assay secondary AB against human IgG, IgM, or IgA (Euro- immun, Lübeck, Germany) [2, 25]. Mouse serum was Functional studies were conducted with sera following analyzed using the same assay with secondary AB against ammonium-sulfate precipitation of immunoglobulins [28] mouse IgG, IgM, or IgA (M31001, A-31570, A-21042; and dialysis (Slide-A-Lyzer® Mini Dialysis Units, 10,000 Thermo Fisher, Rockford, USA). MWCO Plus Float, Thermo Fisher). To assess AB func- tionality, human IPSC-derived neurons were exposed to NMDAR1-AB IgM screening in monkey samples dialyzed serum [10]. For each species, arbitrarily selected seronegative (N = 1) and seropositive samples (N = 2–3) HEK293T cells (50,000) cultured at 37 °C/8% CO2 in were analyzed. Briefly, cells were precooled on ice and DMEM (high glucose, Life Technologies, Carlsbad, USA) washed prior to incubation in cold HBSS with 1:50 diluted were seeded on a 35 -mm dish, grown for 3 days, and dialyzed sera, control NMDAR1-AB (M68-SYSY), or transfected with 3 µg of myc-His-tagged GluN1-1b cloned HBSS alone (negative control) for 30 min/4 oC. After into pcDNA4/TO/myc-His A (Invitrogen, Carlsbad, USA) washing to remove unbound AB, neurons were returned to using Metafectene-Pro (Biontex, Munich, Germany) [10]. their media and incubated for 20 min at 37 oC (three cov- One day post transfection, cells were split onto five poly-D- erslips/sample, endocytosis) or 4 °C (one coverslip/sample, lysine-coated coverslips in a 35 -mm dish and 1 day later, endocytosis control). The remaining surface NMDAR1 was they were fixed with 5% paraformaldehyde (PFA) for 20 exposed to mouse anti-human NMDAR1-AB (N-terminal; min, washed 5× (PBS), permeabilized with 0.1% Triton X- ab134308; Abcam, Cambridge, UK, 1:100), followed by 100 for 5 min, again washed 5× (PBS), and blocked with labeling with secondary donkey anti-mouse IgG (A10036; 5% normal goat serum (NGS; Sigma-Aldrich, Munich, Life Technologies, AlexaFluor®546, 1:100). Neurons were Germany) for 1 h. After five PBS washes, cells were incu- fixed with ice-cold 4% PFA and double stained with bated with serum and monoclonal mouse anti-myc IgG chicken anti-NeuN-AB (266006; SYSY, 1:500) and sec- (clone 9E10, Hollmann-Lab, Bochum) for 1 h, washed ondary donkey anti-chicken AB (703-546-155; Life Tech- with 10× (PBS), incubated for 1 h with fluorescein-labeled nologies, AlexaFluor®488, 1:250). Nuclei were visualized goat anti-monkey IgM (072-11-031; KPL, Gaithersburg, using DAPI (Sigma-Aldrich, 0.01 µg/ml). After PBS wash, USA) and AlexaFluor®594-labeled goat anti-mouse coverslips were mounted on SuperFrost®-Plus slides with IgG (A11005; Thermo Fisher) secondary AB, and PBS Mowiol mounting media (Sigma-Aldrich). Confocal laser- washed 5×. Cells were mounted in Fluoromount-G scanning microscopy was used to quantify NMDAR1 (Southern Biotech, Birmingham, USA) and analyzed density at the membrane (63× glycerol objective; TCS-SP5 via TCS-SP2-AOBS confocal microscope (63× oil Leica-Microsystems, Mannheim, Germany). From each immersion objective; Leica-Microsystems, Wetzlar, coverslip, Z series of optical sections (0.5 μm apart) cov- Germany). The results were independently assessed by ering the three-dimensional extension of neurons were three investigators. acquired (sequential scanning mode, identical acquisition parameters). FIJI-ImageJ software [29] was used to ran- Protein-A assay domly select NeuN+ cells and determine soma profile. Fluorescence intensity/cell surface area (AlexaFluor546) Human serum (for cross-validating clinical standard pro- was automatically measured as readout of cedure and protein-A method), as well as dog, cat, rat, and NMDAR1 surface expression. After background subtrac- monkey serum were labeled with protein-A from Staphy- tion, the mean intensity for each coverslip was determined lococcus aureus, binding the Fc portion of immunoglobu- and fluorescence intensity ratio (37/4 °C) was calculated. lins of different species [26]. Plasma (50 μl) and 25 μgof FITC-conjugated protein-A (Sigma-Aldrich) were incu- BBB-integrity testing bated for 2 h in the dark at room temperature (RT). The mixture was then diluted to 250 μl (PBS) and unbound BBB integrity of 12-month-old ApoE−/− (N = 5) and FITC–Protein-A was removed using 100- kDa Amicon filter ApoE+/+ (N = 5) mice was determined using two different units (Sartorius, Göttingen, Germany), reconcentrating to fluorescent tracers, Evans blue (50 mg/g body weight) [30] ~50 μl[27]. NMDAR1-AB seropositivity was determined and sodium fluorescein (200 mg/g body weight). A detailed using Euroimmun assay combined with commercial description of this method will be published elsewhere [31]. monoclonal mouse NMDAR1-AB (114011; M68, SYSY, Briefly, for tracer quantification in the brain at 4 h after Göttingen, Germany). Samples showing distinct double intravenous injection in the tail vein, animals were PBS labeling were rated “positive” (>98% consensus of three perfused to remove the circulating tracer. Brains were dis- investigators). sected, immediately frozen on dry ice, weighed, and stored H. Pan et al. at −80 °C. Tissue was lyophilized at −56 °C for 24 h under Baseline and post MK-801 locomotion in the open vacuum of 0.2 mBar (Christ LMC-1-BETA-1-16, Osterode, field Germany). For tracer extraction, hemispheres were incu- bated with shaking in 10 ml formamide/mg brain at 57 °C The open-field apparatus consisted of a gray circular for 24 h. Integrated density of tracer fluorescence was Perspex-arena (120 cm diameter; wall height 25 cm). determined in triplicates on a fluorescent microscope Indirect white light illumination ensured constant light (Observer Z2, Zeiss, Germany), equipped with Axio- intensity of 120 lux in the center. Locomotion was mea- CamMRc3, 1×Camera-Adapter, and ZEN2012 blue-edition sured using automated tracking software (Viewer2-Biob- software, recorded at 10× magnification (Plan-Apochromat serve, Bonn, Germany). ApoE−/− and ApoE+/+ littermates 10×/0.45M27). Tracer concentration was calculated using a received four baseline measurements preimmunization and standard curve and normalized to controls (set to 1). post immunization (15 min each), the last followed by intraperitoneal MK-801 (Dizocilpine-[5S,10R]-(+)-5- Mouse immunization methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine hydrogen maleate; 0.3 μg/10 μl PBS/g Sigma- Mice (12-month-old C57BL/6 littermates: ApoE−/−N = 20 Aldrich). MK-801 is a noncompetitive NMDAR antagonist, and ApoE+/+N = 23; genders balanced) were immunized acting as a use-dependent ion-channel blocker, and known with a mixture of GluN1 extracellular peptides and/or to induce psychosis-like hyperactivity in the open field (loss chicken ovalbumin (Sigma-Aldrich), and emulsified in of inhibition) [37]. Directly post injection, locomotor equal volume of complete Freund’s Adjuvant (Myco- activity in open field was analyzed (4 min intervals), with bacterium tuberculosis H37RA plus incomplete Freund’s the first 4 min defined as reference locomotion to express Adjuvant; Becton-Dickinson, Sparks, USA) at a final con- changes over 120 min as % reference. centration of 1 mg/ml [32]. At the tail base, 50 μg of GluN1 peptides and/or 20 μg of ovalbumin were injected sub- Immunohistochemistry cutaneously (each side one). Mice were anesthetized with Avertin (2,2,2- Enzyme-linked immunosorbent assay (ELISA) Tribromoethanol, Sigma-Aldrich), and transcardially per- fused with 4% PFA/Ringer solution (Braun-Melsungen, Orbital sinus blood of immunized mice was stored as EDTA Germany). Brains were removed, postfixed in 4% PFA plasma at −80 °C. ELISA plates (96 well) were coated with overnight at 4 °C, and incubated in 30% sucrose/PBS for 0.5 μg of GluN1 peptide mixture or 0.2 μg of chicken 2 days at 4 °C. Brains were cryosectioned coronally into ovalbumin in 50 μl PBS/well overnight at 4 °C and blocked 30 µm slices and stored in 25% ethylene glycol and 25% with 5% BSA/PBS (Carl Roth, Karlsruhe, Germany). glycerol/PBS at −20 °C. Frozen sections (three/mouse; Mouse plasma (1:1000 or 1:50,000 5% BSA/PBS rostral hippocampus), mounted on SuperFrost®-Plus slides 50 μl/well) was added for 2 h at RT. The signal was (Thermo Fisher, Waltham, USA), were used for cell quan- amplified with horseradish peroxidase-linked anti-IgG tification. For CD3 staining, sections were microwaved 3× (Sigma-Aldrich), and 3,3′,5,5′-Tetramethylbenzidine as for 4 min in citrate buffer (1 mM, pH 6) and blocked with colorimetric substrate (BD Biosciences, San Jose, USA). 5% normal horse serum (NHS), and 0.5% Triton X-100/ Absorbance at 450 nm was measured by microplate reader PBS for 1 h at RT. Incubation with rat anti-mouse CD3 (Tecan-Trading AG, Männedorf, Switzerland). (MCA1477; BioRad, Hercules, USA; 1:100) diluted in 5% NHS, and 0.5% Triton X-100/PBS was performed for two Basic behavioral screening nights/4 °C, followed by incubation with goat anti-rat AlexaFluor®647 (A-21247; Thermo Fisher, Schwerte, The behavioral test battery was performed as described Germany; 1:1000) diluted in 5% NHS, and 0.5% Triton X- previously [33–36]. Starting at age 5 months, experi- 100/PBS for 2 h at RT. For Iba1, GFAP, CD68, and MHC- mentally naïve ApoE−/− and ApoE+/+ littermates under- II staining, sections were blocked with 5% NGS and/or 5% went (during light phase) tests of anxiety, activity and NHS in 0.5% Triton X-100/PBS for 1 h at RT. Incubation exploratory behavior (elevated plus-maze, open field, with rabbit anti-mouse Iba1 (019-19741; Wako-Chemicals hole-board), motor (rotarod, grip strength) and sensory GmbH, Neuss, Germany; 1:1000), or mouse anti-mouse function (visual cliff, olfaction, hearing, hot plate), GFAP (NCL-GFAP-GA5; Novocastra-Leica, Newcastle sensorimotor gating (prepulse inhibition), pheromone- upon Tyne, UK; 1:500), diluted in 3% NGS or 3% NHS, based social preference, and cognitive performance and 0.5% Triton X-100/PBS, was performed overnight, and (IntelliCage place/reversal learning). Males and females incubation with rat anti-mouse CD68 (MCA1957GA; were tested separately. BioRad GmbH, München, Germany, 1:400) and rat Anti-NMDAR1 autoantibodies across mammals anti-mouse MHC-II (14-5321; eBioscience, San Diego, High seroprevalence of NMDAR1-AB across USA, 1:100) diluted in 3% NGS and 3% NHS, and 0.5% mammalian species Triton X-100/PBS, was performed over two nights, all at 4 ° C. Incubation with secondary antibodies was performed We next analyzed by protein-A method serum samples of with goat anti-rabbit AlexaFluor®555 (A-21428; Thermo dogs, cats, rats, baboons, and rhesus macaques. Strikingly, Fisher; 1:500) diluted in 3% NGS, 0.5% Triton X-100/PBS, all mammalian species, independent of their respective life or donkey anti-rabbit AlexaFluor®488 (A-21206; Thermo expectancy, show high NMDAR1-AB seropositivity Fisher, 1:500) or donkey anti-mouse AlexaFluor488 (Fig. 1b). Mouse samples were analyzed using specificAB (A21202; Thermo Fisher, 1:500) or goat anti-rat Alexa- against murine IgA, IgM, and IgG. As known for humans Fluor®647 (A-21247; Thermo Fisher, 1:500), diluted in [6], NMDAR1-AB of the IgG class were the rarest. For 3% NGS or 3% NHS, and 0.5% Triton X-100/PBS for another cross-validation, all monkey samples (N = 100) 1.5 h at RT. Nuclei were counterstained with DAPI (Sigma- were analyzed in blinded fashion by an independent lab Aldrich, 0.01 µg/ml) and sections were mounted using (Bochum; using specific anti-monkey IgM). IgM-positive Aqua-Poly/Mount (Polysciences, Warrington, USA). results coincided with the protein-A positivity by >97% Tile scans of hippocampus were acquired using Leica- (76 of 78). The fraction of protein-A positive but IgM- DMI6000 epifluorescence microscope (20× objective; negative monkey samples (total 22%) likely presents Leica) and Iba1+ and CD3+ cells were counted using NMDAR1-AB of IgA class and IgG class where specific cell counter plug-in of FIJI-ImageJ software [29]. AB were not available. GFAP+ cells in the hippocampus were quantified densito- metrically upon uniform thresholding (expressed as % Age-dependent NMDAR1-AB seroprevalence except respective area). for nonhuman primates and human migrants
Statistical analyses All species revealed age dependence of NMDAR1-AB seroprevalence (χ2 test; dogs: χ2(1) = 11.5, p = 0.01; cats: Statistical analyses were performed using SPSSv.17 (IBM- χ2(1) = 4.8, p = 0.03; rats: χ2(1) = 9.5, p = 0.002; and mice: Deutschland-GmbH, Munich, Germany) or Prism4 Fisher’s exact test p = 0.032) as for humans [5, 8] with the (GraphPad Software, San Diego, California, USA). Group exception of baboons (χ2(1) = 1.0, p = 0.3), where already differences in categorical and continuous variables were >50% of young animals were seropositive. This surprising assessed using χ2, Mann–Whitney U, or Student's t-tests result made us investigate another monkey species, rhesus depending on data distribution/variance homogeneity. macaques, showing again high seroprevalence in old and ANOVA was employed as indicated in display item young animals (χ2(1) = 0.2, p = 0.6) (Fig. 1b). We won- legends. All p-values are two tailed; significance is set to p dered what the difference between humans, dogs, cats, < 0.05; data are presented as mean ± S.E.M. mice, and rats, on one hand, and monkeys, on the other hand, could be, leading to loss of the usual age pattern regarding seroprevalence. Postulating that captivity/non- Results domestication of young monkeys might induce chronic life stress due to maladaptation to the environment, we inves- Cross-validation of NMDAR1-AB detection methods tigated in a hypothesis-driven way whether young human migrants would display a similar increase in NMDAR1-AB To determine NMDAR1-AB seropositivity in mammals seropositivity. Of the GRAS data collection, detailed other than humans, we had to validate the protein-A information on migration was available in a subsample of N detection method [27]. For that, N = 72 paired human = 970 individuals. While nonmigrants show the typical age serum and ventricular CSF samples, prospectively collected association of NMDAR1-AB seroprevalence (χ2(1) = 10.7, from random neurosurgical patients, were analyzed by the p = 0.001), migrants do not (χ2(1) = 0.6, p = 0.4) (Fig. 1c). usual cell-based assay, employing specific secondary AB Seroprevalence in young migrants is significantly higher as for all Ig classes. A total of N = 5 sera turned out compared to young nonmigrants (χ2(1) = 5.381, p = 0.020). NMDAR1-AB positive (titers ≤ 1:100; 3× IgM; 2× IgA; In both monkey species and migrants, the IgM fraction still 0× IgG). Ventricular CSF samples were all negative. For follows the expected age trend, while IgA seems to account cross-validation of NMDAR1-AB of the IgG class, we used for the early increase in NMDAR1-AB seroprevalence serum of a seropositive stroke patient [8]. Application of (Fig. 1c). Presentation of NMDAR1-AB by Ig class in the protein-A method combined with double labeling for extended GRAS sample (N = 4933), with NMDAR1-AB M68 confirmed positive and negative N = 4632 of likely nonmigrants (available information less results (Fig. 1a). detailed) and N = 301 known migrants, illustrates the H. Pan et al.
a NMDAR1-AB SCREENING IgA IgM IgG Gl N transf uN1 AB Step 1 GCLASS
-I Human Protein-A e ct
NTI NMDAR1-AB ed – FITC A DAPI
Step 2 ce l ls (HE A-FITC Serum K29 & ventricular CSF EIN pairs 3T OT )
R ProteinA-FITC P M68 DAPI
b NMDAR1-AB SEROPREVALENCE + c NMDAR1-AB Human Homo sapiens Dogs# Cats# Rats# Mice+ Baboons* Rhesus* Raus Mus Papio Macaca Non-Migrants Migrants Canis familiaris Felis catus norvegicus musculus hamadryas mulaa p=0.4 80 All Ig classes p=0.002 p=0.020
(%) 20 p (%) IgA&IgG p=0.3 p=0.6 p=0.001 =0.4 + 60 + IgM p=0.032 15 0.4 p=0.01 p=0.03 17 AB 1 40 5 14 10 5
39 10 AR1- 1 8 11 20 15 5 3 AB NMDAR1- 4 30 1337 22 69 0 14 13 25 32 38 NMD 26 0 0 18 Age <5y 11-18y <2y 12-22y <2m 14-27m <1.5m 9-33m <3y 7-28y <3y 10-24y Age <35y ≥35y <35y ≥35y (N) 45 98 30 41 18 36 12 21 23 20 28 29 (N) 289 493 100 88
e NMDAR1-AB FUNCTIONALITY Seronegave Seroposive samples d COURSE by Ig class Non-Migrants MigrantsAge-dependent(N=301) (N=4632) 1.5 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 30 25 All Ig classes %)
° C 25 …foesruoc ( 15 +
° /4 1.0 20 5 AB
ce intensity IgM 15 25 35 45 55 65 75
ls 15 R1- ns 0.5 o
DA 10 ratio 37
gs IgA boo 5 Do Cats Rats Mice Ba Rhesus Contr M68 HBSS NM
Fluorescen 0 IgG Neurons 150 289 150 300 100 215 150 296 150 257 150 82 99 152 0 (N) 1 2 1 2 1 2 1 2 1 3 1 2 2 2 Age(y) 13 23 33 43 53 63 73 83 93 Fig. 1 NMDAR1-AB seropositivity and functionality across mam- mammals for all Ig classes combined (#protein-A–FITC/Euroimmun) malian species. a Cross-validation of assays: paired serum and intra- or for individual classes (+Euroimmun; *protein-A–FITC/Euroimmun ventricular CSF samples from neurosurgical patients were tested using and cross-validation with Euroimmun/monkey IgM) presented in the a HEK293T cell-based clinical standard assay for NMDAR1-AB bars; color codes used for consistency and kept also in c and d; age seropositivity (Euroimmun biochip). For step 1, fluorescently labeled given in months (m) or years (y); χ2 or Fisher’s exact test. c NMDAR- IgA-specific, IgM-specific, and IgG-specific secondary AB were used; AB seropositivity of subjects with migration (first and second gen- for method cross-validation (step 2), NMDAR1-AB seropositive and eration) vs. nonmigration history (GRAS data collection); all Ig classes seronegative samples of each Ig class from step 1 were labeled with presented; age split at 35 years; χ2 test. d NMDAR1-AB course by Ig protein-A–FITC conjugate and tested for colocalization (yellow) of classes in serum over age groups in migrants vs. nonmigrants of the protein-A–FITC+ (green) and M68+ (monoclonal mouse NMDAR1- extended GRAS data collection. Note the different course particularly AB followed by Alexa555 donkey anti-mouse IgG red). Representa- for IgA. eFunctionality testing of NMDAR1-AB in human IPSC- tive pictures of both methods using the same seropositive samples derived cortical neurons: degree of internalization expressed as a ratio (IgA, IgM, and IgG) are displayed on the right: upper row step 1/lower of fluorescence intensity measured at 37 and 4 °C; number of neurons row step 2. b NMDAR-AB seropositivity (%) of young and old and sera (N) given; Mann–Whitney U test abnormal course of IgA vs. IgM/IgG seroprevalence over sera provoked NMDAR1 internalization, verifying func- age in migrants (Fig. 1d). tionality (Mann–Whitney U; all p < 0.001) (Fig. 1e).
Functionality of NMDAR1-AB from different BBB dysfunction but normal behavior of mammalian species ApoE−/− mice
To assess whether NMDAR1-AB of the tested species are We next induced endogenous NMDAR1-AB formation in a functional, our endocytosis assay using IPSC-derived mouse model of BBB dysfunction, ApoE−/− mice vs. WT human cortical neurons [10] was employed. All positive littermates, ApoE+/+. Before that, we confirmed in 12- Anti-NMDAR1 autoantibodies across mammals
a DEMONSTRATION OF BBB-LEAKINESS IN ApoE-/- MICE Tracer-extravasaon ApoE-/- EB NaFl
+/+ issue) ApoE t p p Lyophiliza on <0.001 <0.001 EB & NaFl 100 5
%) 80 4 ApoE-/- 4h PBS 60 3 perfusion Formamide ater ( ApoE+/+ w 40 2 er (μg/g brain in
20 c 1
ra 55 5555 B 0 Tra 0 b EXPERIMENTAL OUTLINE GluN1 pepdes Ovalbumin MK-801 Brain & blood collecon OPEN FIELD OPEN FIELD OPEN FIELD Pre-immune serum IMMUNIZATION OPEN FIELD
Days -7-8 03571 10 427 28 c IMMUNIZATION: PEPTIDES AND EFFICACY GluN1 GluN2B Ovalbumin immunizaon (IgG) GluN1 immunizaon (IgG) P1 5 Diluon 1:1000 5 Diluon 1:1000 ApoE+/+ ApoE-/- ApoE+/+ ApoE-/- 4 (N=8) (N=8) 4 (N=8) (N=8) P2 3 3 P3 2 2 P4 1 1 Extracellular 0 0 Optical density (450 nm) Membrane Pre- 35 10 28 Pre- 35 10 28 immune immune Time (days) Time (days)
d OPEN FIELD ACTIVITY OF ApoE+/+ AND ApoE-/- MICE UPON MK-801 INJECTION
ApoE+/+ Ovalbumin (N=11) ApoE-/- Ovalbumin (N=8) ApoE+/+ Ovalbumin+GluN1 (N=12) ApoE-/- Ovalbumin+GluN1 (N=12) 600 F(1,17)=0.2; p=0.7 F(1,22)=5.6; p=0.028 500 400 eli ne
as 300
%B 200 100 0 4 28 52 76 100 120 4 28 52 76 100 120 Time (minutes) Time (minutes)
e INFLAMMATION READOUTS 400 10 F(3,18)=0.3; p=0.8 F(3,18)=0.4; p=0.8 2 2
m 300 /m s 200
cells/mm 5 cell + +
100 CD3 Iba1 5 5 6 6 Iba1 DAPI CD3 DAPI 0 0 5 5 6 6 Fig. 2 Behavioral and morphological effects of endogenous NMDAR1- 801 injection on activity in the open field; results presented as % change AB of the IgG class in a mouse model with open BBB. a Demonstration from baseline (first 4 min post MK-801 set to 100%); no difference in of BBB leakiness in ApoE−/− mice using an intravenously injected MK-801-induced hyperactivity between genotypes after ovalbumin mixture of Evans blue (EB) and sodium fluorescein (NaFl): After brain immunization (one-way repeated measures ANOVA: treatment × group cryopreservation/lyophilization, tracers were extracted with formamide interaction: F(1,17) = 0.2; p = 0.7); increase in hyperactivity (during rise, and quantified; Student’s t-test; b Experimental outline; c Immunization: plateau, decline, and after-effect phases) upon MK-801 in ApoE−/− but Left: GluN1 peptides (P1–P4) located in the extracellular part of the not ApoE+/+ mice immunized against GluN1 (one-way repeated mea- receptor were used for immunization (compare Fig. 3); middle and right: sures ANOVA: treatment × group interaction: F(1,22) = 5.6; p = 0.028). Time course of anti-ovalbumin and anti-GluN1-AB (IgG) upon e Quantification of Iba1+ and CD3+ cells in the hippocampus to assess immunization in ApoE−/− and ApoE+/+ mice; optical density at dilution inflammation in the brain; one-way ANOVA; representative pictures of 1:1000 shown; titers after day 10 reach up to 1:50,000; d Effect of MK- Iba1 (left) and CD3 (right) stainings in the middle H. Pan et al.
Table 1 Basic behavioral screening of male and female ApoE+/+ and ApoE-/- mice
Males Females
Behavioral paradigms Age ApoE+/+ ApoE–/– p-value Age ApoE+/+ ApoE–/– p-value (month) (N) (N) (month) (N) (N)
Anxiety and activity Elevated plus-maze 5 12.6±3.2 19.5±4.0 p=0.14 5 17.5±2.9 14.8±1.3 p=0.98 (time open [%]) (10) (10) U=30.0 (13) (11) U=71.0 Exploratory behavior Hole-board (holes visited [#]) 5 15.2±2.3 11.9±1.9 p=0.30 5 15.5±1.8 15.6±2.9 p=0.96 (10) (10) t(18)=1.07 (13) (13) t(22)=0.96 Open-field Locomotion [m] 5 31.8±1.7 32.7±1.5 p=0.70 5 42.7±1.3 43.7±3.2 p=0.76 (10) (10) t(18)=0.39 (13) (13) t(22)=0.31 Motor learning and coordination Rotarod day 1 (latency to 6 89.3±11.6 130.0±15.3 p=0.06 5 130.9±14.0 133.3±16.0 p=0.91 fall [s]) (10) (10) t(18)=2.01 (13) (11) t(22)=0.11 Rotarod day 2 (latency to 6 140.3±9.4 145.6±17.8 p=0.81 5 179.0±16.8 160.5±19.9 p=0.5 fall [s]) (10) (10) t(18)=0.25 (13) (11) t(22)=0.69 Muscle strength Grip-strength [au] 6 110.2±5.4 122.0±5.0 p=0.15 6 108.8±3.0 115.1±4.4 p=0.26 (10) (10) t(18)=1.52 (13) (11) t(22)=1.16 Heat/pain perception Hot-plate (latency to lick [s]) 5 12.8±0.4 11.9±0.7 p=0.22 5 13.7±0.5 12.4±0.5 p=0.15 (10) (10) t(18)=1.26 (12) (10) t(20)=1.5 Vision Visual-cliff (time on 5 26.5±7.2 22.0±5.6 p=0.85 5 21.7±5.1 29.0±3.9 p=0.13 "air" side [%]) (10) (10) U=47.0 (13) (11) U=45.0 Olfaction Buried food-test (latency to find 5 59.4±9.2 50.6±8.5 (9) p=0.52 5 47.8±12.9 50.7±10.7 p=0.87 cookie [s]) (10) t(17)=0.66 (12) (11) t(21)=0.16 Hearing Acoustic startle at 65dB [AU] 6 0.5±0.04 0.5±0.04 p=0.53 8 0.4±0.1 0.5±0.04 p=0.19 (10) (10) F(1,18)=0.42 (13) (11) F(1,22)=1.82 Acoustic startle at 120dB [AU] 4.5±1.0 (10) 4.8±1.0 (10) 3.3±0.5 4.2±0.6 (13) (11) Sensorimotor gating Mean pre-pulse inhibition [%] 6 44.8±6.7 40.6±7.4 p=0.69 8 57.7±4.1 50.4±6.3 p=0.35 (10) (10) F(1,18)=0.16 (13) (11) F(1,22)=0.91 Pheromone-based social preference Time spent in pheromone 15 1213±50.8 1115±83.7 p=0.33 box [s] (12) (12) t(22)=1.0 Time spent in control box [s] 780.5±75.4 751.1±83.5 p=0.84 (12) (12) t(22)=0.21 Cognitive performance in IntelliCage Place-learning [% target corner 15 34.2±1.3 34.2±1.8 p=0.76 visits]a (12) (13) U=72.0 Reversal-learning [% target 34.2±1.3 34.2±1.8 p=0.17 corner visits]a (12) (13) U=52.0 aas previously described in Netrakanti et al. 2015 Note: All data in the table are mean ± S.E.M.
month-old mice (age of immunization) BBB leakiness using −10.66, p < 0.001; NaFl: t(8) = −8.97, p < 0.001) two fluorescent tracers. While brain water content was (Fig. 2a). We wondered whether this compromised similar in both genotypes, pointing against inflammation, BBB would by itself lead to behavioral abnormalities in ApoE−/− mice showed increased tracer extravasation, con- ApoE−/− mice. A comprehensive behavioral battery, firming BBB dysfunction (Student’s t-test: EB: t(8) = including tests for anxiety, activity, exploratory behavior, Anti-NMDAR1 autoantibodies across mammals motor and sensory function, sensorimotor gating, CD68 and MHCII, was essentially negative and identical pheromone-based social preference, and cognitive perfor- across groups. Moreover, staining for GFAP did not mance did not reveal any differences between genotypes reveal any appreciable density increase in the (Table 1). hippocampus, and thus no sign of astrogliosis (data not shown). Immunization of ApoE−/− and ApoE+/+ mice against NMDAR1-peptides Discussion To explore whether endogenously formed NMDAR1-AB would lead to measurable behavioral and morphological The present work demonstrates high seroprevalence of effects, we immunized 12-month-old ApoE−/− and ApoE+/+ functional NMDAR1-AB of all Ig classes across mammals, littermates against four peptides of the extracellular indicating that these AB are part of a pre-existing auto- NMDAR1/GluN1-domain (including NTD-G7; N368/ immune repertoire [11–17]. As in humans, NMDAR1-AB G369) and ovalbumin or against ovalbumin alone as of the IgG class are the least frequent [6, 20]. The age immunization control (Fig. 2b–c). GluN1 shows >99% related up to >50% NMDAR1-AB seropositivity is inde- sequence homology among all here-tested mammalian pendent of the respective species’ life expectancy, indicating species, with immunizing peptides being 100% homologous that the aging process itself rather than years of exposure to (Fig. 3). Immunization led to high circulating levels of a certain environment triggers NMDAR1-AB formation. specific IgG (titers up to 1:50,000). Efficacy of immuniza- However, our knowledge on predisposing factors and tion and time course of IgG appearance as determined by inducing mechanisms is limited. Specific autoimmune- ELISA were comparable for NMDAR1-peptides and oval- reactive B cells may get repeatedly boosted by, e.g., bumin across genotypes, making a simple boosting effect of infections, neoplasms, or the microbiome, and less effi- NMDAR1-peptides on pre-existing NMDAR1-specificB ciently suppressed over a lifespan likely owing to a gradual cell clones rather improbable (Fig. 2c). loss of immune tolerance upon aging [14]. Unexpectedly, we find the age-dependence lost in non- − − Psychosis-related behavior of ApoE / mice upon human primates and in human migrants that all display an MK-801 challenge early-life rise in NMDAR1-AB seropositivity, mainly of IgA. The intriguing possibility that chronic life stress, Open-field tests measuring baseline preimmunization and known to be present in human migrants [38] and animals in postimmunization locomotion did not reveal any differences captivity [39], acts as a trigger of early NMDAR1-AB between genotypes and/or immunization groups (Fig. 2b; formation is worth pursuing experimentally in the future. A not shown). After 4 weeks, the endogenously formed large proportion of migrants in our human samples are NMDAR1-AB of the IgG class induced strong hyper- suffering from neuropsychiatric illness. This may addi- activity (psychosis-like symptoms [37]) upon MK-801 tionally support our chronic stress hypothesis since migra- challenge in ApoE−/− mice only. In contrast, ApoE+/+ tion is recognized as an environmental stressor predisposing mice behaved comparably to ovalbumin-only immunized to mental disease [40]. Further studies should screen wild- mice of both genotypes (Fig. 2d; all p > 0.5). Thus, an open life monkeys and species in zoos for NMDAR1-AB. BBB together with sufficiently high titers of AB (to reach a Experimental confirmation of our findings provided, threshold loss of NMDAR1 surface expression) is a pre- NMDAR1-AB (IgA) may even serve as stress markers. In requisite for the observed behavioral perturbation upon fact, earlier reports show that total serum-Ig of all classes, MK-801. most prominently IgA, respond to psychological stress [41]. NMDAR1-AB might thus belong to a set of stress-boosted No inflammation in hippocampus of immunized AB. Interestingly, we also find accumulated seroprevalence − − ApoE / and ApoE+/+ mice of 23 other brain-directed AB [6] in young migrants vs. nonmigrants increased (data not shown), suggesting a glo- Immunohistochemistry did not show any evidence of bal inducer role of chronic stress in humoral autoimmunity. inflammation in either genotype and/or immunization Earlier work has shown that AB against brain antigens in group. Numbers of Iba1+ and CD3+ cells as markers of general are common among mammals [42], but no study microglia and T cells, respectively, were comparable for has so far systematically screened nonhuman mammals for total hippocampus (one-way ANOVA: Iba1: F(3,18) = 0.3; NMDAR1-AB. As an exception, a recent report described p = 0.8; CD3: F(3,18) = 0.4; p = 0.8) (Fig. 2e) and for all “anti-NMDAR1 encephalitis” in the young polar bear Knut hippocampal subfields separately (all p-values > 0.2; not [27]. Based on the present findings, Knut may have shown). Also, staining for microglial activity markers, belonged to those nondomesticated species in captivity— H. Pan et al.
Fig. 3 Alignment of GluN1-1b receptor amino acid sequence across all dimensional presentation in Fig. 2c) and nonhomologous amino acids mammalian species tested. Regions containing the four peptide in pink. SP signal peptide, S1, S2 segments of the ligand-binding sequences (peptides 1–4: P1: AA35–53, P2: AA361–376, P3: domain, TMD A transmembrane domain A, TMD B transmembrane AA385–399, and P4: AA660–811) used in the immunization experi- domain B, TMD C transmembrane domain C ment are highlighted in yellow and light brown (compare three- Anti-NMDAR1 autoantibodies across mammals comparable to monkey species investigated here—that are EXTRABRAIN EU-FP7, and the Niedersachsen-Research Network affected by chronic early-life stress, inducing NMDAR1- on Neuroinfectiology (N-RENNT). The authors thank all subjects AB seropositivity. Pre-existing NMDAR1-AB of this bear for participating in the study, and the many colleagues who have contributed over the past decade to the extended GRAS data may have ultimately shaped the clinical picture of an collection. encephalitis of unexplained origin (likely infectious according to the zoo’s pathology reports) where an epileptic Author contributions Concept, design, and supervision of the study: seizure led to drowning [27]. HE; Data acquisition/analysis/interpretation: HP, BO, ED, DT, MM, JS, JW, DW, CKS, AR, KS, RT, KMR, StB, YAK, HM, MB, WS, This interpretation is supported by our novel auto- GS, FJK, RM, SB, KAN, JKK, MH, FL, and HE; Drafting manuscript: immune model, namely, mice immunized against HE, with the help of BO and HP; Drafting display items: HE and BO, NMDAR1-peptides. Even high titers of endogenously with the help of HP, MM, DT, and JS. All authors read and approved fi formed NMDAR1-AB (IgG; up to 1:50,000) that induce the nal version of the manuscript. psychosis-like behavior upon MK-801 challenge in − − Compliance with ethical standards ApoE / mice, with here-confirmed open BBB, do not lead to any appreciable signs of encephalitis. This dissociation of Conflict of interest WS is a member of the board and holds stocks in behavioral/symptomatic consequences and inflammation in Euroimmun AG. HM is a full-time employee of Synaptic Systems the brain is of major importance for clinicians [14]. For GmbH. The remaining authors declare that they have no conflict of instance, earlier studies reported an influence of NMDAR1- interest. AB infusions into the hippocampus on learning and mem- Open Access This article is licensed under a Creative Commons ory in mice [43], and others found increased NMDAR1-AB Attribution-NonCommercial-NoDerivatives 4.0 International License, seroprevalence in patients with mild cognitive impairment which permits any non-commercial use, sharing, distribution and and Alzheimer's disease [44, 45]. However, while all natu- reproduction in any medium or format, as long as you give appropriate rally occurring NMDAR1-AB that have pathogenic potential credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this irrespective of epitope and Ig class [10], and upon entry to license to share adapted material derived from this article or parts of it. the brain (or via intrathecal production) can shape brain The images or other third party material in this article are included in functions in the sense of NMDAR antagonism, only a frac- the article’s Creative Commons license, unless indicated otherwise in a ’ tion of individuals happens to have underlying encephalitis credit line to the material. If material is not included in the article s Creative Commons license and your intended use is not permitted by of various etiologies, which is then called anti-NMDAR statutory regulation or exceeds the permitted use, you will need to encephalitis. The highly variable neuropathology and obtain permission directly from the copyright holder. To view a copy response to immunosuppression of this condition [2, 3, 46] of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/. may point to a broad range of possible encephalitogenic mechanisms (from infection to oncology or genetics) which References need to be diagnosed and specifically treated [14]. Even though it is unclear how NMDAR1-AB are 1. Dalmau J, Tuzun E, Wu HY, Masjuan J, Rossi JE, Voloschin A, generated by chronic stress, it should be considered that et al. Paraneoplastic anti-N-methyl-D-aspartate receptor encepha- NMDAR1 are not only expressed in the brain but also by litis associated with ovarian teratoma. Ann Neurol. peripheral organs and tissues, including adrenal glands 2007;61:25–36. 2. Dalmau J, Gleichman AJ, Hughes EG, Rossi JE, Peng X, Lai M, and gut [47] which may be involved in triggering et al. Anti-NMDA-receptor encephalitis: case series and analysis NMDAR1-AB formation but may also be functionally of the effects of antibodies. 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Affiliations
1 1 2 1 3 1 1 Hong Pan ● Bárbara Oliveira ● Gesine Saher ● Ekrem Dere ● Daniel Tapken ● Marina Mitjans ● Jan Seidel ● 1 1 3 1 4 Janina Wesolowski ● Debia Wakhloo ● Christina Klein-Schmidt ● Anja Ronnenberg ● Kerstin Schwabe ● 3 5 2 4 6 1 Ralf Trippe ● Kerstin Mätz-Rensing ● Stefan Berghoff ● Yazeed Al-Krinawe ● Henrik Martens ● Martin Begemann ● 7 5 8 9 2,10 Winfried Stöcker ● Franz-Josef Kaup ● Reinhard Mischke ● Susann Boretius ● Klaus-Armin Nave ● 4 3 11 1,10 Joachim K. Krauss ● Michael Hollmann ● Fred Lühder ● Hannelore Ehrenreich
1 Clinical Neuroscience, Max Planck Institute of Experimental 7 fi Medicine, Göttingen, Germany Institute for Experimental Immunology, af liated to Euroimmun, Lübeck, Germany 2 Department of Neurogenetics, Max Planck Institute of 8 Experimental Medicine, Göttingen, Germany Small Animal Clinic, University of Veterinary Medicine, Hannover, Germany 3 Department of Biochemistry I—Receptor Biochemistry, Ruhr 9 University, Bochum, Germany Functional Imaging Laboratory, Leibniz Institute for Primate Research, Göttingen, Germany 4 Department of Neurosurgery, Hannover Medical School, 10 Hannover, Germany DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany 5 Department of Pathology, Leibniz Institute for Primate Research, 11 Göttingen, Germany Department of Neuroimmunology, Institute for Multiple Sclerosis Research and Hertie Foundation, University Medicine Göttingen, 6 Synaptic Systems GmbH, Göttingen, Germany Göttingen, Germany
5. PROJECT III
5. PROJECT III – Excitation-inhibition dysbalance as predictor of autistic phenotypes
Overview of Project III
The phenotypic variability observed in autism defines it as a spectrum disorder with three main groups: autism, Asperger’s syndrome and pervasive developmental disorder not otherwise specified, with all of them sharing deficits in social communication/interaction, restricted interests and stereotypic/repetitive behavior (Buxbaum et al. 2013). However, autistic phenotypes transcend diagnostic categories. Schizophrenia is among the many neurodevelopmental diseases with autistic symptoms contributing to disease severity (Kastner et al. 2015, Stepniak et al. 2015). Additionally, subthreshold deficits of the core phenotypic domains of autism spectrum disorders (ASD) can be found in the general population in different levels, supporting the continuous nature of autistic behaviors (Jones et al. 2013, Kastner et al. 2015).
Involvement of synaptic and synapse regulating genes is a constant observation across ASD genomic screenings as well as in monogenic forms of autism. This suggests a central role for defects in synaptic structure and function in the pathogenesis of ASD. Additionally, it has been postulated that hyperexcitability of cortical circuits, due to an increased ratio between excitation and inhibition (E/I), might be associated with ASD. This imbalance in E/I can be related with increased excitatory glutamatergic signalling, or with reduction in inhibition due to a reduction in GABAergic signalling (Rubenstein et al. 2003, Nelson et al. 2015). Furthermore, evidence of a reduced E/I ratio, as consequence of a shifted E/I ratio favouring inhibition over excitation, has also been reported in both mouse and in vitro human IPS neurons (Dani et al. 2005, Marchetto et al. 2010). Altogether, several genetic mouse models of ASD point to altered E/I ratio as a neural mechanism underlying the pathophysiology of ASD. Human studies also support the notion that an imbalanced E/I ratio represents a convergence point for many of the genetic causes associated with ASD. In fact, cellular abnormalities involving neural transmission might be associated with neurotransmitter generation, release, reception and re-uptake. Quantification of neurotransmitter levels provided some suggestive evidence supporting the involvement of altered E/I in ASD. Increased serum levels of glutamate and GABA have been reported in ASD when compared to neurotypical individuals. Magnetic resonance spectroscopy studies provided additional information by measuring the brain levels of GABA, glutamate and the glutamate precursor glutamine. Nevertheless, the results are very heterogeneous across studies and do not allow firm conclusions. Indirect evidence for imbalanced E/I ratio has been provided by
85 PROJECT III
electroencephalography and magnetoencephalography data. However, they do not point a clear direction of such imbalance (Dickinson et al. 2016).
Considering the transcending nature of autistic phenotypes, the severity of autistic traits in a population of schizophrenic patients was evaluated. To cover the ASD diagnostic domains of difficulties in social interaction, communication, and repetitive and stereotypic behaviour in the GRAS population, an autism severity score (PAUSS) based on specific items of the Positive and Negative Syndrome Scale (PANSS) was employed. The PANSS is a standardized clinical observation tool, applied to assess positive, negative and psychopathology symptom severity in schizophrenia (Kay et al. 1987, Kastner et al. 2015). By ranking individuals according to their PAUSS score it was possible to categorize them in two main groups: low and high PAUSS, in which higher scores represent a higher severity of autistic traits.
Male individuals of both groups were then selected and matched for age, handedness and antipsychotic medication to access their E/I ratio using TMS. Non-invasive brain stimulation techniques as TMS allow probing cortical excitability in vivo (Hallett 2007). An input-output (I/O) curve to assess excitability and a paired-pulse TMS protocol to monitor intracortical inhibition and facilitation were applied in schizophrenic patients presenting contrasting PAUSS scores. Threshold or suprathreshold stimulation of the motor cortex with TMS activates cortical neurons via the induction of electric currents, generating motor evoked potentials (MEP) in the respective target muscle. MEP amplitude serves as a measure of cortical excitability. Therefore, the minimum TMS intensity to elicit an MEP (motor threshold) and the I/O curve, in which the MEP response due to increasing stimulation intensity is monitored, are the global measures of corticospinal excitability. These readouts depend largely on the activation of voltage-gated ion channels, with the probable exception of MEPs resulting from high TMS intensities, in which the glutamatergic system is also involved (Paulus et al. 2008). In contrast, paired-pulse TMS protocols allow the relative specific determination of intracortical excitability. Here, a first sub-threshold TMS stimulus, which does not elicit an MEP and whose effects are consequently intracortical, is followed by a suprathreshold stimulus. The alteration of the MEP amplitude elicited by the second stimulus depends on the interstimulus interval and could serve as index of intracortical excitability. Short latency intracortical inhibition probes primarily inhibitory GABAergic interneurons, whereas intracortical facilitation probes glutamatergic excitatory connections (Kujirai et al. 1993, Paulus et al. 2008). While no differences in resting or active motor thresholds were observed between the two phenotypic groups, the group of schizophrenic patients with more severe autistic features showed higher cortico-spinal excitability and higher intracortical inhibition compared to the group with low autistic features. In contrast, intracortical facilitation
86 PROJECT III did not differ between groups. Additionally, the E/I ratio and the severity of autistic traits were positively correlated.
Our results suggest that the severity of autistic traits in these patients couples with altered GABAergic neurotransmission and activity of voltage-gated ion channels, with no clear changes in glutamatergic neurotransmission. Enhanced corticospinal excitability could have been driven by altered voltage-gated ion channel activity or glutamate signalling, particularly at high TMS intensities (Paulus et al. 2008). Since glutamate-driven intracortical facilitation did not differ between groups, a predominant impact on voltage-gated ion channels is more likely. Overall, the results of this study support the concept of a disturbed E/I balance in subjects with autistic features, which is based on altered GABAergic and voltage-gated ion channel functions.
These findings support the pathophysiological continuum of autistic traits and the convergence of the associated phenotypic and functional aspects in a common pathway that transcends diagnostic criteria.
Original publication
Oliveira B*, Mitjans M*, Nitsche MA, Kuo M-F and Ehrenreich H. Excitation-inhibition dysbalance as predictor of autistic phenotypes. Under revision.
*Authors with equal contribution
Personal contribution: I actively participated in outlining and writing the manuscript, together with my supervisor. I was significantly involved in the data analysis, prepared the figures and figure legends and contributed to the overall preparation of the manuscript. Additionally, I was responsible for the submission phase and will actively participate in the revision process.
The manuscript of Project III is currently under revision and is presented in the appendix section of the thesis.
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6. SUMMARY AND CONCLUSIONS
6. SUMMARY AND CONCLUSIONS
The complexity of neuropsychiatric conditions reflects the heterogeneity of their etiological factors, in which a combination of genetic and environmental risk factors shapes phenotypes. However, one must dissect single contributors to disease and health status having in mind the possibility of overlapping phenotypes across diseases, and that an individual’s biological repertoire influences their response to environmental triggers. The present thesis focused on the molecular and functional characterization of autoantibodies targeting the NMDAR and their role beyond its pathognomonic association with anti-NMDAR encephalitis (Project I and II); and how the severity of autistic traits in schizophrenic patients relates with imbalances in excitation and inhibition (Project III).
The nervous and immune systems integrate environmental cues that shape their function and influence the dynamic interaction between both systems. Alterations in this delicate balance are emerging as strong contributors to brain disease. Autoantibodies reactive with several brain expressed proteins have been associated with neurological conditions, either as an etiological factor or by contributing to disease severity (Crisp et al. 2016). The presence of different NMDAR-AB isotypes has been previously described in healthy individuals and individuals suffering from other conditions that do not include anti-NMDAR encephalitis (Dahm et al. 2014, Finke et al. 2017). However, the functional and molecular properties of these autoantibodies remained to be characterized. Projects I and II focused on understanding the molecular and functional underpinnings of NMDAR-AB and the role of the BBB in preventing the antagonizing effects of these autoantibodies in the brain.
In project I, a detailed characterization of different NMDAR-AB isotypes, that included IgM, IgA and IgG provided information regarding their functional effect on NMDAR cellular location and glutamate evoked-responses. Furthermore, a systematic mapping of the NMDAR epitopes recognized by these autoantibodies was performed. Altogether, this information broadened the spectrum of action of these autoantibodies. Anti-NMDAR encephalitis research has been focusing mainly on the effects of NMDAR-AB of IgG isotypes (Dalmau et al. 2008, Gleichman et al. 2012, Moscato et al. 2014). This work demonstrated that other isotypes can antagonize NMDAR activity and potentially impact brain function upon access to the brain. Beyond the pathogenic context of anti-NMDAR encephalitis, these NMDAR-AB can induce NMDAR antagonism and consequently alter glutamatergic signalling. In vivo, exposure to NMDAR-AB leads to extracellular accumulation of glutamate and the consequent hyperglutamatergic state might dysregulate the excitability of neuronal networks via excitotoxic neuronal dysfunction (Manto et al. 2010). Post mortem studies of anti-
91 SUMMARY AND CONCLUSIONS
NMDAR encephalitis patients did not reveal any pathological evidence of neuro-axonal injury (Bien et al. 2012). Thus, it is plausible to think that the NMDAR-AB glutamate-mediated excitotoxicity does not reach the necessary threshold to induce neurodegeneration but it is enough to alter glutamatergic neurotransmission. Indeed, in rat hippocampal neurons NMDAR-AB (IgG) from CSF of schizophrenic patients alter the surface dynamics and nanoscale organization of synaptic NMDAR, preventing long-term potentiation at glutamatergic synapses (Jezequel et al. 2017). The polyspecificity and polyclonality, of several of these NMDAR-AB, along with their functionality, contrasts with the relevance credited exclusively to one epitope (G7 site of the NTD) in the context of anti-NMDAR encephalitis and suggests a more diverse interaction of these autoantibodies with NMDAR.
Project II demonstrated that NMDAR-AB are also present in other mammal species and, as in humans, its seroprevalence increases with age (Hammer et al. 2014). However, of all the species tested two monkey species presented high seroprevalence in young age. This was also observed in humans that underwent migration. Altogether, this data broadened the number of species with reported seropositivity for NMDAR-AB and raised some interesting questions about the environmental triggers of antibody production such as chronic life stress, that warrant further clarification. Furthermore, using a novel autoimmune mouse model, with endogenous production of NMDAR-AB, the BBB emerged as a central player in avoiding the antagonizing effects of these autoantibodies in the brain. For the first time, in physiological conditions of NMDAR-AB production, it was shown that the BBB has a protective effect regarding the antagonizing action of these autoantibodies in the brain. NMDAR-AB are able to bind to several brain areas (Castillo-Gomez et al. 2016). An intact BBB prevents NMDAR- AB from reaching the brain parenchyma by restricting immunoglobulins influx and performing active efflux of the low levels of immunoglobulins that cross the BBB in physiological conditions. Additionally, it tightly controls the transendothelial migration of B cells, which once in the brain parenchyma could contact with NMDAR and, in the case of a secondary antigen encounter, be boosted to produce NMDAR-AB intrathecally (Diamond et al. 2009). In the presence of a compromised BBB, such as in ApoE-/- mice, endogenous production of NMDAR-AB leads to an increased sensitivity to MK-801 treatment translated to an exaggerated hyperactive behaviour in the open field due to a cumulative antagonizing effect of these autoantibodies and MK-801. Moreover, the presence of NMDAR-AB in the brain per se does not trigger an inflammatory reaction which points to the necessity of other contributors for the development of an encephalitogenic phenotype.
The data gathered in Project I and II broadens the spectrum of action of these autoantibodies by attributing functional roles to other isotypes, and consolidates the protective role of the BBB in seropositive individuals. NMDAR-AB emerge as components of the immune repertoire with the potential to shape brain function and possibly contribute to psychiatric
92 SUMMARY AND CONCLUSIONS phenotypes (Jezequel et al. 2017). It provides relevant information for the clinical management of asymptomatic individuals seropositive for NMDAR-AB and the importance of monitoring BBB integrity in these individuals (Ehrenreich 2017).
Project III addresses the continuous nature of autistic phenotypes in a sample of schizophrenic male individuals and demonstrates how imbalances in excitation and inhibition can correlate with the severity of autistic traits. By combining readouts of sociability, communication and stereotypic behaviour, in a single dimension, it was possible to classify schizophrenic patients regarding the severity of their autistic traits. Furthermore, non-invasive assessment of cortico-spinal excitability and intracortical inhibition using TMS provided information on excitation and inhibition levels in these individuals. The results of this project suggest a positive correlation between the severity of autistic traits and imbalances in E/I ratio in schizophrenic patients, mainly mediated by altered GABAergic and voltage-gated ion channel functions. It supports previous reports of altered GABAergic neurotransmission in schizophrenia, adding a new correlation with phenotypic readouts (Wobrock et al. 2008).
To conclude, this thesis comprises translational work in clinical neuroscience. It broadens the current knowledge on neuro-immune interactions, focusing on NMDAR-AB (patho)physiology, and on functional readouts associated with phenotypic differences within schizophrenia. Ultimately, it can contribute to improve clinical practices for diagnostics and disease management.
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8. APPENDIX
APPENDIX
8. APPENDIX
Accepted co-author publications
Manuscript 1 Oliveira B & Ehrenreich H. Pursuing functional connectivity in NMDAR1 autoantibody carriers. Letter to Lancet Psychiatry. 2018; 5(1):21-22
Personal contribution: I actively participated in the discussion of the original publication that this letter refers to, after presenting the paper in the context of a lab journal club. I wrote the manuscript together with my supervisor, Prof. Dr. Dr. Hannelore Ehrenreich.
Manuscript 2 Mitjans M*, Begemann M*, Ju A*, Dere E, Wüstefeld L, Hofer S, Hassouna I, Balkenhol J, Oliveira B, Van der Auwera S, Tammer R, Hammerschmidt K, Völzke H, Homuth G, Cecconi F, Chowdhury K, Grabe H, Frahm J, Boretius S, Dandekar T, Ehrenreich H. Sexual dimorphism of AMBRA1 related autistic features in human and mouse. Translational Psychiatry. 2017; 10;7(10):e1247.
Personal contribution: I contributed to the design and execution of the experiments to determine AMBRA1 expression levels in individuals with different genotypes of interest. Additionally, I prepared and analysed brain sections of aged-matched mice regarding the MRI experiments for quantification of inhibitory cells in the hippocampus (unpublished data). I was involved in the preparation of the manuscript.
Manuscript 3 Ehrenreich H, Castillo-Gomez E, Oliveira B, Ott C, Steiner J, Weissenborn K. Circulating NMDAR1 autoantibodies of different immunoglobulin classes modulates evolution of lesion size in acute ischemic stroke. Neurology Psychiatry and Brain Research. 2016; 22(1).
Personal contribution: I differentiated the human IPS cell-derived neurons from patient fibroblasts, standardized and performed the NMDAR1 internalization assay to test functionality of human autoantibodies targeting the NR1 subunit of the aforementioned receptor. Additionally, I contributed to the study design and the overall writing of the manuscript.
113 APPENDIX
Manuscript 4 Ott C, Martens H, Hassouna I, Oliveira B, Erck C, Zafeiriou MP, Peteri UK, Hesse D, Gerhart S, Altas B, Kolbow T, Stadler H, Kawabe H, Zimmermann WH, Nave KA, Schulz- Schaeffer W, Jahn O, Ehrenreich H. Widespread expression of erythropoietin receptor in brain and its induction by injury. Molecular Medicine. 2015; 21:803-815.
Personal contribution: I prepared the human IPS cell lines used to test EPOR antibody and performed the immunocytochemistry experiments. Additionally, I validated the detection of EPOR in UT-7 cells using the same antibody by flow cytometry. I contributed to the overall preparation of the manuscript.
114 APPENDIX - Manuscript 1 Correspondence
https://doi.org/10.1016/2215-0366(17)30465-0
a gambling disorder is confronted with Psychiatry, Academic Medical Center, University of any conclusion linking the described EGMs. A more detailed understanding Amsterdam, Amsterdam, Netherlands (RJvH); and connectivity disturbance to NMDAR1 Gambling and Social Determinants Unit, School of of the interactions between these Public Health and Preventive Medicine, Monash autoantibodies is difficult based on machine design features and University, Melbourne, VIC, Australia (CL) these data which lack the adequate aspects of human decision-making 1 Schull ND. Addiction by design: machine control. Only a well-matched control and behaviours, including their gambling in Las Vegas. Princeton: Princeton group of patients with encephalitis University Press, 2012. interactions within vulnerable 2 Harris A, Griffiths MD. A critical review of the without history of NMDAR1 groups (adolescents, those with a harm-minimisation tools available for autoantibody positivity could allow any mental illness, or under substantial electronic gambling. J Gambling Stud 2017; speculation in this direction. Healthy 33: 187–221. psychosocial distress), will provide 3 Harrigan KA, Dixon M. PAR sheets, individuals are not a proper control. valuable insights for producing safer probabilities, and slot machine play: Although information on functional implications for problem and non-problem gambling products. The use of virtual gambling. J Gambling Issues 2009; 23: 81–110. connectivity in age-matched and reality and computational or decision 4 Breen RB, Zimmerman M. Rapid onset of gender-matched healthy individuals neuroscience approaches can provide pathological gambling in machine gamblers. provides some baseline for comparison, J Gambling Studies 2002; 18: 31–43. ecologically valid and real-time 5 Robinson TE, Berridge KC. The neural basis of they do not allow dissecting the investigations of affective, cognitive, drug craving: an incentive-sensitization theory effects of NMDAR1 autoantibodies and physiological changes while of addiction. Brain Res Brain Res Rev 1993; on brain connectivity. A proper 18: 247–91. gambling. control population would require the Urgent reform of EGM regulations underlying inflammatory context of to limit the impact of structural an encephalitic brain without NMDAR1 characteristics on gambling-related Pursuing functional autoantibodies. A dysconnectivity harm is needed. Opportunities abound connectivity in NMDAR1 syndrome can be expected in any kind of for regulatory attention to reduce the encephalitis.2–4 In addition, the absence prevalence and harm of gambling, autoantibody carriers of NMDAR1 autoantibodies at the including venue and machine time of fMRI in a considerable number accessibility, modification of EGM We read with interest the article by of individuals questions an ongoing structural characteristics, enhanced Michael Peer and colleagues.1 The influence of them on functional user understanding and information, authors investigated by resting-state connectivity. Long-term persisting and use of systems to assist users to functional MRI (fMRI) a reasonable effects of NMDAR1 autoantibodies make and observe limits to gambling.2 number of patients (n=43; 24 of in their absence (ie, once they are The time has come to prevent further which were reported previously) who eliminated by immunosuppression, damage associated with gambling and were diagnosed earlier with so-called plasmapheresis, or other means) have protect our communities. anti-NMDAR encephalitis (treatment not yet been documented anywhere. No funding was received in relation to the present regimens were not mentioned). It They would also be difficult to prove article. MY reports grants from the National Health is acknowledged that a collection of non-experimentally. On a side note, and Medical Research Council, Australian Research patients with encephalitis who are catatonia is still classified as a positive Council, The David Winston Turner Endowment Fund, from Monash University, and from law firms also NMDAR1 autoantibody-positive symptom. in relation to expert witness report or statement. is not easy to obtain. Of this collection, Future studies evaluating the AC reports grants from the National Health and a large proportion had, on the day of importance of autoantibodies for brain Medical Research Council, during the conduct of the study. CL reports grants from the Victorian fMRI, negative NMDAR1 autoantibody functions should employ resting-state Responsible Gambling Foundation, Australian titres. In fact, only 17 of 43 patients fMRI for standardised comparative Research Council, City of Melbourne, Maribyrnong were CSF positive and 27 of 43 were assessment of different forms of acute City Council, City of Whittlesea, Alliance for Gambling Reform, outside the submitted work. seropositive, if we consider a titre of and chronic encephalitides, including RJvH and KH declare no competing interests. 1:10 as a reasonable cut-off. Resting- encephalitis with autoantibodies state fMRI was done at highly variable directed against brain epitopes, like *Murat YÜcel, Adrian Carter, Kevin timepoints after the initial diagnosis NMDAR1 autoantibodies. In addition, Harrigan, Ruth J van Holst, Charles and led to the authors’ conclusion of a NMDAR1 autoantibody carriers (all Ig Livingstone “characteristic pattern of whole-brain classes)5 with a compromised blood– [email protected] functional connectivity alterations in brain barrier should be investigated Brain and Mental Health Laboratory, Monash anti-NMDAR encephalitis that is well using this method. Institute of Cognitive and Clinical Neurosciences and School of Psychological Sciences, Monash suited to explain the major clinical We declare no competing interests. symptoms of the disorder”. University, Melbourne, VIC, Australia (MY, AC); Bárbara Oliveira,*Hannelore Ehrenreich Gambling Research Laboratory, University of Undoubtedly, a sexy new method [email protected] Waterloo, ONT, Canada (KH); Department of was applied to a hot topic. However,
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Clinical Neuroscience, Max Planck Institute of Additionally, machine learning treatment response, and follow-up. Experimental Medicine, Hermann-Rein-Strasse 3, analyses based on resting-state fMRI Indeed, this should include NMDAR 37075 Goettingen, Germany data reliably distinguished patients antibody carriers of IgG, IgA, and IgM 1 Peer M, Prüss H, Ben-Dayan I, Paul F, Arzy S, Finke C. Functional connectivity of large-scale from controls. antibodies, given their association brain networks in patients with anti-NMDA Notably, 86% of our patients were with cognitive impairments.6 receptor encephalitis: an observational study. positive for IgG NMDAR antibodies at Lancet Psychiatry 2017; 4: 768–74. We declare no competing interests. 2 Kharabian Masouleh S, Herzig S, Klose L, et al. the time of imaging and all patients Functional connectivity alterations in patients had CSF IgG NMDAR antibodies Michael Peer, Harald Prüss, with chronic hepatitis C virus infection: a at diagnosis, the well-accepted Inbal Ben-Dayan, Friedemann Paul, multimodal MRI study. J Viral Hepat 2017; Shahar Arzy, *Carsten Finke 24: 216–25. hallmark of anti-NMDAR encephalitis. 3 Lin Y, Zou QH, Wang J, et al. Localization of The significant resting-state fMRI [email protected] cerebral functional deficits in patients with connectivity disturbances, irrespective Computational Neuropsychiatry Laboratory, non-neuropsychiatric systemic lupus Department of Medical Neurosciences, Hebrew erythematosus. Hum Brain Mapp 2011; of antibody persistence or disease University of Jerusalem Medical School, Jerusalem, 32: 1847–55. duration, indicate that the observed Israel (MP, IB-D, SA); Department of Neurology, 4 Liu Y, Wang H, Duan Y, et al. Functional brain Hadassah Medical Center, Jerusalem, Israel (MP, network alterations in clinically isolated changes reflect long-term effects on IB-D, SA); Department of Neurology (HP, FP, CF), syndrome and multiple sclerosis: a graph- brain activity. Long-term persisting based connectome study. Radiology 2017; Berlin Center for Advanced Neuroimaging Analyses 282: 534–41. deficits in meanwhile antibody- (CF), and NeuroCure Clinical Research Center (FP), 5 Castillo-Gomez E, Oliveira B, Tapken D, et al. negative NMDAR encephalitis are Charité–Universitätsmedizin Berlin, Berlin, All naturally occurring autoantibodies against rather the well-documented rule than Germany; German Center for Neurodegenerative the NMDA receptor subunit NR1 have Diseases, Berlin, Germany (HP); The Rachel and 5 pathogenic potential irrespective of epitope the exception in the literature and Selim Benin, School of Computer Science and and immunoglobulin class. Mol Psychiatry our clinical experience. Engineering, Hebrew University of Jerusalem, 2016; published online Aug 9. DOI:10.1038/ Jerusalem, Israel (IB-D); Experimental and Clinical mp.2016.125. We respectfully disagree that Research Center, Max Delbrueck Center for healthy controls are not an adequate Molecular Medicine and Charité– control group. A comparison with Universitätsmedizin Berlin, Berlin, Germany (FP); Authors’ reply carefully matched healthy individuals and Berlin School of Mind and Brain, Humboldt- Universität zu Berlin, Berlin, Germany (CF) We thank Bárbara Oliveira and is not only common practice 1 Peer M, Prüss H, Ben-Dayan I, Paul F, Arzy S, Hannelore Ehrenreich for their in computational neuropsychiatry Finke C. Functional connectivity of large-scale comments on our work.1 Resting- and functional neuroimaging, it is brain networks in patients with anti-NMDA receptor encephalitis: an observational study. state functional MRI (fMRI) is indeed essential to identify disturbances of Lancet Psychiatry 2017; 4: 768–74. a promising new imaging tool brain activity. Other autoimmune 2 Stam CJ. Modern network science of that already provided substantial CNS disorders differ in their level of neurological disorders. Nat Rev Neurosci 2014; 15: 683–95. insight into the pathophysiology of inflammation, the pathophysiological 3 Woodward ND, Cascio CJ. Resting-state several neuropsychiatric disorders, mechanisms, and affected brain functional connectivity in psychiatric including major depressive disorder, regions, and therefore cannot provide disorders. JAMA Psychiatry 2015; 72: 743–44. 4 Finke C, Kopp U a, Pajkert A, et al. Structural schizophrenia, and Alzheimer’s a meaningful “inflammatory baseline”. hippocampal damage following disease.2,3 Using resting-state By contrast, preliminary analyses show anti-N-methyl-D-aspartate receptor fMRI, we identified a characteristic that resting-state fMRI can distinguish encephalitis. Biol Psychiatry 2016; 79: 727–34. 5 Titulaer MJ, McCracken L, Gabilondo I, et al. pattern of functional connectivity characteristic connectivity alterations Treatment and prognostic factors for long-term alterations in the largest cohort of between anti-NMDAR encephalitis outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study. anti-NMDA receptor encephalitis and other autoimmune encephalitides Lancet Neurol 2013; 12: 157–65. examined with MRI so far. These such as anti-LGI1 encephalitis. We 6 Finke C, Bartels F, Lütt A, Prüss H, Harms L. connectivity disturbances correlated therefore believe that resting-state High prevalence of neuronal surface autoantibodies associated with cognitive with symptoms of psychosis and fMRI can become a highly useful tool deficits in cancer patients. J Neurol 2017; memory impairment and extend in the work-up of neuropsychiatric 264: 1968–77. recent observations of hippocampal patients, potentially able to damage and white matter alterations.4 facilitate the differential diagnosis,
22 www.thelancet.com/psychiatry Vol 5 January 2018 116 OPEN Citation: Transl Psychiatry (2017) 7, e1247; doi:10.1038/tp.2017.213 www.nature.com/tp
ORIGINAL ARTICLE Sexual dimorphism of AMBRA1-related autistic features in human and mouse
M Mitjans1,2,14, M Begemann1,2,3,14,AJu1,14, E Dere1,2, L Wüstefeld1, S Hofer4, I Hassouna1, J Balkenhol5, B Oliveira1, S van der Auwera6, R Tammer4, K Hammerschmidt7, H Völzke8, G Homuth9, F Cecconi10,11, K Chowdhury12, H Grabe6, J Frahm4, S Boretius13, T Dandekar5 and H Ehrenreich1,2
Ambra1 is linked to autophagy and neurodevelopment. Heterozygous Ambra1 deficiency induces autism-like behavior in a sexually dimorphic manner. Extraordinarily, autistic features are seen in female mice only, combined with stronger Ambra1 protein reduction in brain compared to males. However, significance of AMBRA1 for autistic phenotypes in humans and, apart from behavior, for other autism-typical features, namely early brain enlargement or increased seizure propensity, has remained unexplored. Here we show in two independent human samples that a single normal AMBRA1 genotype, the intronic SNP rs3802890-AA, is associated with autistic features in women, who also display lower AMBRA1 mRNA expression in peripheral blood mononuclear cells relative to female GG carriers. Located within a non-coding RNA, likely relevant for mRNA and protein interaction, rs3802890 (A versus G allele) may affect its stability through modification of folding, as predicted by in silico analysis. Searching for further autism-relevant characteristics in Ambra1+/ − mice, we observe reduced interest of female but not male mutants regarding pheromone signals of the respective other gender in the social intellicage set-up. Moreover, altered pentylentetrazol-induced seizure propensity, an in vivo readout of neuronal excitation–inhibition dysbalance, becomes obvious exclusively in female mutants. Magnetic resonance imaging reveals mild prepubertal brain enlargement in both genders, uncoupling enhanced brain dimensions from the primarily female expression of all other autistic phenotypes investigated here. These data support a role of AMBRA1/ Ambra1 partial loss-of-function genotypes for female autistic traits. Moreover, they suggest Ambra1 heterozygous mice as a novel multifaceted and construct-valid genetic mouse model for female autism.
Translational Psychiatry (2017) 7, e1247; doi:10.1038/tp.2017.213; published online 10 October 2017
INTRODUCTION on monogenetic grounds, have helped in approaching the 17,18 Autism spectrum disorders (ASD) are extremely heterogeneous underlying common biology. neurodevelopmental conditions, affecting ~ 1% of the population. The estimated heritability of autism approximates 90%. We Typical, shared symptoms range from social communication and note, however, that monogenetic forms including copy number o interaction deficits, including decreased attraction by and variations altogether account for 20%, leaving ~ 80% of cases unexplained, which also enter the final common pathway of compromised reading of social signals, restricted interests, – disease expression.1 3 Importantly, normal genetic variants, mainly repetitive behaviors or pronounced routines, to reduced cognitive – – single nucleotide polymorphisms (SNPs), likely contribute to the flexibility.1 4 Early brain enlargement5 8 and predisposition to 9,10 manifestation of autistic phenotypes. This is indicated by the epileptic seizures are among the reported non-behavioral results of genome-wide association (GWAS) and respective characteristics found in this disorder category. Causes likely polygenic-risk studies on autism,2,19,20 but even more so by converge at the synapse, as indicated by mutations of synaptic phenotype-based genetic association studies (PGAS), reporting an genes or by mutations causing quantitative alterations in synaptic accumulation of ‘unfortunate’ normal variants, so-called pro- protein expression, half-life or degradation, and are reflected by a autistic genotypes, to be associated with increasing severity of virtually autism-pathognomonic neuronal excitation–inhibition autistic traits.21–23 In fact, phenotypical continua of autistic – dysbalance.4,11 15 Neuroligin-4 mutations, for instance, belong to features from health to disease suggest underlying mechanisms the most common causes of monogenetic heritable autism.16 of quantitative rather than qualitative nature.21,24 Genetic Construct-valid and face-valid mouse models of autism, building modifiers, like protective genes and environmental (co-)factors,
1Department of Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany; 2DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany; 3Department of Psychiatry and Psychotherapy, UMG, Georg-August-University, Göttingen, Germany; 4Biomedizinische NMR Forschungs GmbH, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; 5Department of Bioinformatics, Biocenter, University of Würzburg, Würzburg, Germany; 6Department of Psychiatry and Psychotherapy, University Medicine, and German Center for Neurodegenerative Diseases (DZNE) Greifswald, Greifswald, Germany; 7Cognitive Ethology Laboratory, German Primate Center, Göttingen, Germany; 8Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany; 9Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany; 10IRCCS Fondazione Santa Lucia and Department of Biology, University of Rome Tor Vergata, Rome, Italy; 11Unit of Cell Stress and Survival, Danish Cancer Society Research Center, Copenhagen, Denmark; 12Department of Molecular Cell Biology, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany and 13Department of Functional Imaging, German Primate Center, Leibniz Institute of Primate Research, Göttingen, Germany. Correspondence: Professor H Ehrenreich, Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Hermann-Rein-Str. 3, Göttingen 37075, Germany. E-mail: [email protected] 14These authors contributed equally to this work. Received 13 June 2017; revised 1 August 2017; accepted 17 August 2017 Sexual dimorphism of AMBRA1-associated autistic traits M Mitjans et al 2 mainly those acting during uterine and early postnatal develop- longitudinal aspects of the diagnosis as well.39,40 GRAS, complying with ment may also modulate autism severity.25,26 Helsinki Declaration, was approved by Ethics Committees of Georg-August- ASD has an overall gender distribution of ~ 4:1 males/ University, Göttingen, Germany, and participating centers. All study females.1,2,27 Remarkably, little research has focused on the participants (European-Caucasian 95.6%; other 1.8%; unknown 2.6%) and, reasons for this disparity. Better understanding of this difference, if applicable, legal representatives gave written informed consent. Of the however, could lead to major advancements in the prevention or 1105 successfully genotyped patients, 66.7% were male (N = 737), 33.3% 28 female (N = 368), aged 39.46 ± 12.58 years (range: 17–79). For genetic case– treatment of ASD in both genders. We previously reported that control analysis, healthy GRAS blood donors were employed,39 in total an Ambra1 (activating molecule in Beclin1-regulated autophagy) N = 1258 (European-Caucasian 97.8%; other 2%; unknown 0.2%), 61.6% partial loss-of-function genotype is associated with the autism-like male (N = 775), 38.4% female (N = 483), aged 37.45 ± 13.21 (range: 18–69) behavior in female mice. The restriction to the female gender of years. Voluntary blood donors widely fulfill health criteria according autism generation by a defined genetic trait has thus far remained to the national guidelines for blood donation, ensured by a broad pre- unique.29 Ambra1 is a positive regulator of a principal player in donation screening process containing standardized questionnaires, interviews, hemoglobin, blood pressure, pulse and body temperature autophagosome formation, Beclin1. Importantly, autophagy has 39 already been linked to autism in recent work.30,31 Moreover, determinations. Ambra1 is involved in other developmentally relevant processes in the nervous system and in neuronal function.32,33 Although Replication: population-based cohort (SHIP-O). The general population sample comprises N = 2359 homozygous subjects, mean age 49.8 ± 16 homozygosity of the Ambra1 null mutation causes embryonic (range = 20–81) years, N = 1144 males, N = 1215 females, from baseline lethality, heterozygous mice with reduced Ambra1 expression examinations of Study of Health in Pomerania (SHIP), approved by the 33 appear completely normal at first view. Only upon comprehen- Ethics Committee, University Greifswald, and conducted in North-East sive behavioral characterization, a striking autism-like phenotype Germany.41 of Ambra1+/ − females emerges. This trait is quantifiable by the autism severity composite score, which even allows a behavior- Phenotyping. For quantification of autistic phenotypes, we used the based genotype predictability of 490%.29,34 As first mechanistic Positive and Negative Syndrome Scale (PANSS)42-based autism severity 24 hint explaining the prominent gender difference, stronger score (PAUSS) with slight modifications (Figure 1a), available for 1067 reduction of Ambra1 protein in the cortex of Ambra1+/ − females patients. For the replication sample, the Instrumental Support Index (ISI) 29 was taken as proxy, indicating quality of instrumental and emotional was found. 43 23 support, expected to be low in autistic individuals. It was cross- Until now, no association of AMBRA1 genotypes with autistic validated with PAUSS in GRAS subjects, with social support operationalized features has been described in humans, therefore still questioning as self-reported number of individuals a person can rely on in case of 29 the construct-validity of our mouse model. However, a recent emergency.23 For both measures of social support (intercorrelation 0.77), GWAS on schizophrenia identified a genetic risk variation on higher score values (z-transformed; range: 1.5–9) represent higher social chromosome 11 (11p11.2) in a region containing AMBRA1.35 support, that is, lower autistic features. Schizophrenia and ASD show considerable syndromic overlap, including deficits in social cognition and communication,24,36 and Genotyping. GRAS subjects were genotyped using semi-custom Axiom at least a subgroup of schizophrenia is also regarded as a disorder MyDesign Genotyping Array (Affymetrix, Santa Clara, CA, USA), based on a of the synapse.37 CEU (Caucasian residents of European ancestry from UT, USA) marker The present study has therefore been designed to explore backbone, including 518 722 SNPs, plus custom marker-set of 102 537 SNPs. Genotyping was performed by Affymetrix on a GeneTitan platform whether any autism-relevant phenotype association with normal with high quality (SNP call rate 497%, Fisher’s linear-discriminant, AMBRA1 genotypes would emerge in humans, thereby supporting heterozygous cluster-strength offset, homozygote-ratio offset).23,44,45 +/ − the construct-validity of our Ambra1 mouse model. In addition, Markers were selected according to our SOP for PGAS23 using following we aimed at defining potential further characteristics of ASD in selection criteria: (1) SNPs in Hardy–Weinberg equilibrium; (2) SNPs with − Ambra1+/ mice, namely (1) decreased interest in social odors, as minor allele frequency (MAF ⩾ 0.2) allowing for statistical analyses; (3) SNPs highly relevant social signals in mice, (2) increased epileptic not in high linkage disequilibrium (LD) with other selected SNPs (r2 o0.8). predisposition as in vivo readout of neuronal excitation–inhibition Based hereon, only rs3802890-A/G remained for analysis. dysbalance and (3) early brain enlargement, as recognized in SHIP-0 subjects were genotyped using Affymetrix Genome-Wide SNP Array-6.0 (genotyping efficiency 98.6%). Imputation of genotypes was human autism. Indeed, we show here that also in humans, an 46 performed with software IMPUTE v0.5.0 against 1000-Genomes (pha- AMBRA1 genotype, the intronic SNP rs3802890-AA, located in a se1v3) reference-panel using 869 224 genotyped SNPs.41 Rs3802890 was long non-coding (lnc) RNA, is linked to autistic features and imputed with IQ = 1. characterized by partial loss-of-function in females. Moreover, we +/ − demonstrate in Ambra1 mice prepubertal brain enlargement. In silico analyses. Genome sequences were established according to latest Only in female mutants, we see loss of interest in sex pheromones available releases (human-genome vs32–2015; mouse-genome 2016). and altered seizure propensity. Iterative sensitive sequence comparisons were conducted47 and evaluated including detailed genome and transcriptome mapping. Expression of the rs3802890-containing RNA region was derived from latest largest MATERIALS AND METHODS collection of ESTs available at NIH.48 LncRNA matches were also ‘ established according to latest human lncRNA release at NCBI. RNA In all experiments, the experimenters were unaware of genotypes ( fully 49 blinded’). folding used RNAfold. For demonstrating rs3802890-A/G differences, thermodynamic ensemble structures drawing encoded base-pair prob- abilities were used. Protein binding regions were calculated using Human studies RNAanalyzer50 and CatRapid,51 coding potential was calculated using Discovery: schizophrenia subjects and healthy controls (GRAS). The Göttin- Genscan.52 gen Research Association for Schizophrenia (GRAS) data collection consists of 41200 deep-phenotyped patients, diagnosed with schizophrenia or AMBRA1 and EST TCAAP2E6309 mRNA expression. Peripheral blood schizoaffective disorder (DSMIV-TR38), recruited across Germany since mononuclear cells (PBMC) were isolated from morning blood, obtained 2005.39,40 Diagnosis is based on a comprehensive examination, lasting for via phlebotomy into CPDA-vials (Citrate-Phosphate-Dextrose-Adenine, at least 4 h (the examination often took much longer, with breaks in Sarstedt, Germany), applying standard Ficoll-Paque-Plus isolation proce- between, dependent on the patient’s condition). It is guided by the GRAS dure (GE-Healthcare, Munich, Germany). Total RNA extraction was done manual, which contains standardized interviews, psychopathology and using miRNeasy Mini-kit (Qiagen, Hilden, Germany). For reverse transcrip- neuropsychology testing. Moreover, careful study of all the medical tion, 1 μg of cDNA was applied using a mixture of oligo(dT)/hexamers, discharge letters and charts of every single individual aids in assessing dNTPs, DTT and 200U SuperscriptIII (Life Technologies, Darmstadt,
Translational Psychiatry (2017), 1 – 9 Sexual dimorphism of AMBRA1-associated autistic traits M Mitjans et al 3
Figure 1. Human AMBRA1-rs3802890 G/A: association with autistic features. (a) Quantification of autistic symptoms using PAUSS (PANSS Autism Severity Score.24 Note the high intercorrelation of PAUSS items and the high internal consistency of the scale (Spearman rank correlation coefficients; Cronbach’s α). (b) PGAS using AMBRA1-rs3802890 and PAUSS score: female AA subjects display a higher PAUSS score than GG subjects in the discovery sample; mean ± s.e.m.; two-tailed Mann–Whitney U-test (data-corrected by linear regression analysis for age). (c) Trends of positive association between rs3802890-AA genotype and sub-items of PAUSS, more pronounced in females; mean ± s.e.m.; two- tailed Mann–Whitney U-test. (d) The highly significant correlation of PAUSS and social support underlines the validity of social support as an autism proxy phenotype; mean ± s.e.m. (e) Genotype effect of AMBRA1-rs3802890 on degree of social support in the discovery sample, again significant in females; mean ± s.e.m.; Mann–Whitney U-test. (f) Replication of the genotype and gender effect of AMBRA1-rs3802890 using social support as proxy in the general population; linear regression analyses (bootstrap; data-corrected for age); mean ± s.e.m. (g) Relative AMBRA1 mRNA expression in peripheral blood mononuclear cells (PBMC) is reduced in female AA (risk SNP) carriers: shown is the AMBRA1 mRNA expression in AA carriers, given as mean Δ-value compared to GG carriers (GG males N = 33; GG females, N = 14). Values of individual AA males (N = 35) and AA females (N = 33) are expressed in %GG (mean) of the respective gender. The Δ-value is calculated as: Δ = %GG–100%; mean ± s.e.m.; two-tailed Mann–Whitney U-test.
Germany). AMBRA1 RNA expression was measured using quantitative real- Mouse line and housing. Ambra1 mutation in mice is caused by a time PCR. The cDNA was diluted 1:25 in 10 μl reaction-mix, containing 5 μl truncated, non-functional Ambra1 protein via insertion of a gene-trapping of SYBR-green (Life Technologies) and 1pmol/primer: vector into the murine Ambra1 gene.33 Ambra1 wild-type (WT, Ambra1+/+) − AMBRA1-Fw: 5′-GACCACCCAATTTACCCAGA-3′ and heterozygous (Ambra1+/ ) littermates of both genders with 499% − AMBRA1-Rv: 5′-GATCATCCTCTGGGCGTAGTA-3′ C57BL/6 N background were used (male Ambra1+/ × female WT-C57BL/ GAPDH-Fw: 5′-CTGACTTCAACAGCGACACC-3′ 6N). Genotyping was performed as described.29 Males and females were GAPDH-Rv: 5′-TGCTGTAGCCAAATTCGTTGT-3′ kept in separate ventilated cabinets (Scantainers; Scanbur Karlslunde, Technical triplicates were run on LightCycler480 (Roche-Diagnostics, Denmark), group-housed, with woodchip bedding and nesting material, Mannheim, Germany). Relative AMBRA1 expression was calculated using 12 h-light-dark cycle, 20–22 °C, food/water ad libitum. the threshold-cycle method (LightCycler480 Software1.5.0SP3-Roche) and normalization to GAPDH. EST TCAAP2E6309 RNA expression was measured Social intellicage paradigm. For pheromone-based social preference test, − using traditional PCR. Extracted RNA, cDNA synthesized with oligo-dT Ambra1+/+ and Ambra1+/ mice were separately group-housed in large primers with/without hexamers, or genomic DNA were used as template type4 cages after weaning until age 8 weeks. After transponder with the following primers: implantation, they were put into intellicages (IntelliCage; TSE-Systems, EST TCAAP2E6309-Fw: 5′-GGCAGAGCAGAATGGATAGACA-3′ Bad Homburg, Germany), placed inside standard laboratory rodent cages EST TCAAP2E6309-Rv: 5′-AACGCCTGTTATCTGGGATCA-3′ (height 20.5 cm, length 55 cm, width 38.5 cm; Techniplast-Model-2000, Germany) with floor covered by sawdust bedding.53 Each intellicage contains four housing shelters beneath the food hopper. Left and right of Mouse studies the intellicage, two social boxes are connected via plastic tubes, each Investigations were carried out in agreement with guidelines for welfare of equipped with two ring RFID-antennas to track individual mice. IntelliCage experimental animals, issued by the Federal Government of Germany and software records time spent in social boxes. Experiments are performed Max Planck Society, approved by local animal care and use committee during the light phase. After habituation for 1 h to social boxes containing (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsi- fresh bedding, mice undergo the pheromone-based social preference test: cherheit, Oldenburg, Germany). for 1 h they can freely choose between a social box with used bedding of
Translational Psychiatry (2017), 1 – 9 Sexual dimorphism of AMBRA1-associated autistic traits M Mitjans et al 4 C3H mice of opposite gender and another box with only fresh bedding. WT likewise, most PAUSS sub-items show this trend in females but mice typically prefer used bedding containing pheromones. not males (Figures 1b and c).
Magnetic resonance imaging for morphometry. Mice were anesthetized fl fl A role of AMBRA1-rs3802890-AA for female autistic features is (5% iso urane), intubated and kept at 1.75% iso urane/5% oxygen by fi active ventilation with constant respiratory frequency (85 breaths per con rmed in a general population sample minute; Animal-Respirator-Advanced, TSE-Systems). Magnetic resonance Even though this highly targeted approach to an association of imaging (MRI) was performed at 7 and 9.4T (Bruker Biospin MRI, Ettlingen, only one available AMBRA1 SNP with autistic traits in schizophrenic Germany). Radiofrequency excitation and signal reception were accom- individuals was already encouraging, we aimed at replication of plished with use of a birdcage resonator (inner diameter, 72 mm) and a this finding in a general population sample. For this, a social four-channel phased-array surface-coil, respectively. T2-weighted MRI data support score, derived from ISI,43 was used as proxy phenotype, were acquired with three-dimensional fast spin-echo MRI sequence expected to be low in individuals with autistic features.23 Cross- (repetition-time TR = 3.5 s, effective echo-time TEeff = 55 ms, 12 differently validation of social support (operationalized as the self-reported phase-encoded echoes, 56 min measuring time) at isotropic spatial resolution of 100 μm. From these datasets, polygonal surface models number of individuals a person can rely on in case of emergency) of selected brain structures were generated by importing DICOM with PAUSS in the discovery sample (GRAS) yielded a high images into AMIRA (Visage-Imaging, Berlin, Germany). Structures of negative Spearman rank correlation (Figure 1d), underlining the interest (whole brain, hippocampus, cerebellum, olfactory bulb, ventricles) relevance of this proxy for autistic features. Again, the social were manually and semi-automatically labeled with segmentation editor support score disclosed a genotype effect (rs3802890-AA/-GG) in on three-dimensional label fields (80 horizontal, 192 coronal, 144 sagittal both discovery and replication sample, more pronounced in slices). females (Figures 1e and f). Thus, in two independent human cohorts, a single normal variant in the AMBRA1 gene, rs3802890, is Pentylentetrazol-induced seizures. Mouse groups were tested during the associated with autism-related behaviors predominantly in light phase at postnatal day 23 or at 13 months. Seizures were induced by females. single intraperitoneal injection of pentylentetrazol (PTZ) (50 mg kg− 1; Sigma-Aldrich, Taufkirchen, Germany). After injection, mice are observed for 30 min in their home cage.54 Response to PTZ injection is quantified: (1) Consequence of AMBRA1-rs3802890-A versus G on mRNA hypoactivity: decrease in mobility until rest in crouched or prone position, expression in human PBMC and in silico prediction of potential abdomen at bottom; (2) partial clonus (PC): clonic seizure in face, head or underlying mechanisms forelimbs; (3) generalized clonus (GC): sudden loss of upright posture, In some subjects, PBMCs were available for AMBRA1 mRNA whole-body clonus including all limbs and tail, rearing and autonomic analysis. Although female GG (N = 14) versus male GG (N = 33) signs; and (4) tonic-clonic seizure (TC): generalized seizure up to tonic hind- carriers had higher expression levels (0.0059 versus 0.0045 limb extension and death. Latencies to (2)–(4) are used to calculate individual seizure scores (ISS), where factors weight relative severity: AMBRA1/GAPDH; P = 0.05), AA carriers of both genders (males ISS = 1000/(0.2 × PC-latency+0.3 × GC-latency+0.5 × TC-latency).55–57 N = 35; females N = 33) did not differ in their level (males 0.0049; females 0.0046; P = 0.62), which was comparable to that of male GG carriers. Comparing both genders, AMBRA1 mRNA expression Statistical methods in PBMC of AA (risk SNP) relative to GG carriers is reduced in Case-control analysis and test for deviation from Hardy–Weinberg 58 females but not males (P = 0.017; Figure 1g), possibly indicating equilibrium was performed using PLINK1.07. Statistics for human partial loss-of-function of AMBRA1 in AA females. This relative phenotype–genotype associations and mouse studies were conducted with SPSS v.17.0 (IBM-Deutschland, Munich, Germany), STATA MP-v.13.1 reduction found in PBMC of women resembles the situation in cortex of female mice: normal WT females have higher Ambra1 (StataCorp, College Station, TX, USA) and Prism4 (GraphPad-Software, +/ − San Diego, CA, USA). Statistical tests used are always given in figure expression than WT males, whereas Ambra1 females show +/ − legends. Data are presented as mean ± s.e.m., statistical significance was stronger relative Ambra1 reduction compared to Ambra1 set to P = 0.05. males.29 A detailed map of human AMBRA1 gene is explained in Figure 2a. Exploring the location of the intronic rs3802890, a RESULTS similarity to expressed sequence tags (EST) from NCBI database A normal AMBRA1 genotype, rs3802890-AA, is associated with arises (Figure 2a). The predicted RNA folding of the transcribed autistic traits predominantly in female schizophrenic individuals EST TCAAP2E6309, covering the SNP region, is remarkably Only one directly genotyped and—according to our PGAS SOP23 influenced by the presence in rs3802890 of G versus A —suitable SNP, AMBRA1-rs3802890-A/G, was available in our array. (Figure 2b). As we find EST TCAAP2E6309 RNA expressed in PBMC Case–control analysis (1105 schizophrenic versus 1258 healthy and other human tissues (Figure 2c), relevance of this lncRNA for GRAS subjects) yielded comparable genotypic and allelic chi- AMBRA1 mRNA or protein levels may be assumed. square comparison (MAF = 0.31; controls: AA = 607, AG = 532, Together, these data suggest that AMBRA1 likely shapes autistic GG = 119; cases: AA = 507, AG = 505, GG = 93; genotypic: behavior also in humans in a sexually dimorphic way. This across- fi χ2 = 2.974, df = 2, P = 0.226; allelic: χ2 = 0.242, df = 1, P = 0.623). species unique female autism generation by a de ned genetic Thus, rs3802890 is not associated with the schizophrenia trait that appears to cause partial loss-of-function encouraged us to continue searching for further autism-specific readouts in our diagnosis. +/ − Next, PGAS was performed with rs3802890-AA/-GG and Ambra1 mouse model. PAUSS24 as quantitative measure of autistic traits (Figure 1a). In − previous work, we have demonstrated that autistic features cross Pheromone-based social preference is reduced in Ambra1+/ diagnostic borders and can be quantified not only in ASD, but also females in schizophrenia and other diseases as well as in healthy Sex pheromones have an important role in social behavior individuals.21,23,24 For quantification, we developed the PAUSS, a throughout the animal kingdom.59–62 Mice typically favor a social dimensional instrument based on PANSS,42 capturing the context that contains pheromones of the opposite gender. In continuous nature of autistic behaviors.24 PGAS revealed an autistic phenotypes, social interest, approach and communication association: AA carriers display higher PAUSS scores than GG as well as understanding of social signals are compromised.1–4,62 subjects (P = 0.039). Interestingly, when separating genders, the We therefore designed a novel intellicage set-up to test PAUSS association remains significant only for females and, pheromone preference as potential autism-relevant readout in
Translational Psychiatry (2017), 1 – 9 Sexual dimorphism of AMBRA1-associated autistic traits M Mitjans et al 5 a
b c
Figure 2. AMBRA1-rs3802890 G/A: in silico approach to mechanistic insight. (a) Detailed map showing the human AMBRA1 gene (NCBI- accession: 55626; chromosomal location: Chr.11: 46 396 412–46 594 069). The AMBRA1 mRNA (21 exons) is mapped at the bottom. The location of AMBRA1-rs3802890 is indicated by a red asterisk. The region shared between all chromosomes (nucleotides from around 9530 to 11 030 on AMBRA1; red arrow) is indicated. Similar to man, the murine Ambra1 gene region matches on all chromosomes (nucleotides from around 68 110–69 940 on murine Ambra1). However, the chromosomal match region of AMBRA1/Ambra1 is different between both species. Both match regions show similarities to expressed sequence tags (EST) from NCBI database (best match EST gi|22688027 in human and gi|44663783 in mice; both with full-length alignment) and to specific lncRNAs in Refseq database (best match human: Refseq Accession NR_126435.1 named LINC00504; best match in mice: Refseq Accession NR_131899.1 named Mrqpra6). (b) RNA folding of human AMBRA1 transcribed EST comparing the G allele with the A allele. The presentation of thermodynamic ensemble folding stresses differences in the obtained structure. The color code indicates pairing propensities. The fold is further supported by reoccurring similar differences comparing several foldings and also lengths using software mFold. As template for folding, the full RNA sequence of EST from myelogenous leukemia cells (496 nucleotides long, 98% identity; full-length alignment; genbank accession BM149074.1; pediatric acute myelogenous leukemia cell (FAB M1) EST TCAAP2E6309) is shown. This RNA encodes no protein, has no introns/exons and has no complementary match in any other chromosomal region. (c) EST TCAAP2E6309 expression in all tested tissues: RNA was isolated from peripheral blood mononuclear cells (PBMC), human glioblastoma tissue (GB), and human placenta (PL). PCR was performed from cDNA. To exclude false positive results by genomic DNA contamination, several controls were performed (DNase digestion; respective RNA amplification). Genomic DNA from whole blood was used as positive control (+) and ddH2O as negative control (− ).
Ambra1+/ − mice. Upon free choice between a social box contain- Female Ambra1+/ − mice show altered seizure propensity ing used bedding from mice of the opposite gender and another Another frequently observed trait, connected with autistic social box with fresh bedding only, WT male and female and behaviors, not only in syndromic forms of autism, is epileptic − Ambra1+/ male mice behave as expected, namely choose to stay seizures.9,10 Most likely, seizure predisposition reflects the autism- − longer in the respective ‘pheromone box’. In contrast, Ambra1+/ pathognomonic neuronal excitation–inhibition dysbalance.4,11–15 females fail to show this preference (Figure 3). In our Ambra1+/ − model, prepubertal female mutants displayed reduced response to PTZ, namely longer latency to the first whole- MRI analysis reveals brain enlargement in Ambra1+/ − mice body seizure and decreased seizure score compared to female WT (Figure 5b). This early resistance turned into the opposite response Brain enlargement has been described both in children and adults – at older age: Number of tonic-clonic seizures and duration of with ASD.5 8 Recently, brain volume overgrowth in children was fi 7 whole-body seizures were enhanced in 3-months (data not linked to the emergence and severity of autistic social de cits. We shown) and 13-months-old Ambra1+/ − females versus WT, also measured by high-resolution MRI (T2-weighted) brain dimensions +/ − resulting in reduced survival (Figures 5c and d). Male mutants did in Ambra1 versus WT mice. Whole brain and hippocampus not differ from WT at any time point investigated. Together, these were enlarged in male and female mutants at postnatal day 23 +/ − +/ − data support Ambra1 mice as multidimensional model of (around puberty). Cerebellum was increased in female Ambra1 human autism. mice only (Figure 4). Sizes of olfactory bulb and ventricles in Ambra1+/ − mice were similar to WT. Repeated examination of females at age 13 months revealed persistence of the increased DISCUSSION − brain dimensions (Figure 5a). We note that Ambra1+/ mice are We previously reported in female Ambra1+/ − mice a discrete the first autism model showing autism-typical brain enlargement. behavioral trait, reminiscent of human ASD.29 In the present study, Regarding this particular readout, genders were comparable, we extend this finding, showing for we believe the first time uncoupling in this model autism-like behavior (only females) from that AMBRA1 may—in likewise sexually dimorphic manner—be brain dimensions. relevant also in humans for the expression of a female autistic
Translational Psychiatry (2017), 1 – 9 Sexual dimorphism of AMBRA1-associated autistic traits M Mitjans et al 6
Figure 3. Impaired pheromone preference in female Ambra1 mutants. (a) Intellicage apparatus with connected social boxes. (b) Time spent in social boxes with used or fresh bedding or delta difference scores for the indicated genotypes. Upper row females; lower row males; mean ± s. e.m. presented. Within-group comparisons performed with paired t-tests, between-group with Mann–Whitney U-tests; all tests two-tailed.
Figure 4. Brain enlargement in prepubertal Ambra1 mutants of both genders. Shown are results of high-resolution magnetic resonance imaging (MRI) (T2-weighted). Brain regions of interest (whole brain, hippocampus, cerebellum, olfactory bulb, ventricles) in 23day-old female (upper row) and male (lower row) mice of both genotypes are presented; mean ± s.e.m.; two-tailed unpaired t-tests. Right side: Representative pictures of 3D-reconstructed brains of both genotypes illustrate brain enlargement in mutants.
Translational Psychiatry (2017), 1 – 9 Sexual dimorphism of AMBRA1-associated autistic traits M Mitjans et al 7
Figure 5. Persistent brain enlargement and altered pentylentetrazol (PTZ)-induced seizure propensity in female Ambra1 mutants. (a) Brain regions of interest (whole brain, hippocampus, cerebellum, olfactory bulb, ventricles) were analyzed by high-resolution magnetic resonance imaging (MRI) in 13-months-old female Ambra1+/ − and WT mice; mean ± s.e.m.; two-tailed unpaired t-tests. (b) PTZ-induced seizure propensity (intraperitoneal injection of 50 mg kg−1) in 23-day-old WT and Ambra1+/ − mice of both genders; mean ± s.e.m. presented; two-tailed unpaired t-tests. (c) PTZ-induced seizure propensity (intraperitoneal injection of 50 mg kg− 1) in 13-months-old female WT and Ambra1+/ − mice; mean ± s.e.m. presented; two-tailed unpaired t-tests. (d) Survival of PTZ-induced seizures (intraperitoneal injection of 50 mg kg− 1) in 13- months-old female WT and Ambra1+/ − mice; Kaplan–Meier survival analysis. phenotype. The female preponderance, unique thus far in autism brain of neonatal female as compared to male rats.64 In this sense, genetics, may even help illuminating some general molecular AMBRA1/Ambra1 adds to the number of proteins shown to underpinnings of gender susceptibility to brain disease. Remark- underlie sexually dimorphic effects on the brain.65,66 ably, in a highly targeted association approach, using the only In a first in silico search for mechanisms, we saw that the AMBRA1 SNP available for analysis, rs3802890-A/G, we find in two lncRNA, covering the SNP region, shows highly diverse folding independent populations, the GRAS sample of schizophrenic upon presence of G versus A allele. This pronounced structural individuals and the SHIP sample of general population subjects, effect may influence AMBRA1 mRNA and/or protein stability and relevance for this marker regarding autistic traits in women. Partial will be subject for further investigation. − loss-of-function, reflected by a relative decrease in AMBRA1 mRNA Returning to the Ambra1+/ mouse model, we extend our levels in PBMC of female risk genotype (AA) carriers, may suggest earlier findings29 to crucial, additional autistic features, so far not an underlying autism-causing mechanism similar to that in systematically addressed in genetic models of autism, namely heterozygous mice where Ambra1 reduction was stronger in early brain enlargement, altered propensity towards epileptic female than male mutant cortex.29 seizures and reduced pheromone preference. The question of how AMBRA1/Ambra1 reduction may influence Brain enlargement is a consistently reported feature in human – synaptic function, thereby causing the autism-pathognomonic autism, both in adults and children.5 8 Already upon first neuronal excitation–inhibition dysbalance,4,11–15 still remains description of autism, increased size of the head was observed unanswered. We may, however, speculate that reduced autop- in affected children.67 The substrate underlying the enlarged brain − hagy at synaptic terminals,30,31,63 likely more pronounced in has remained obscure, and the Ambra1+/ mouse model may Ambra1+/ − females,29 influences synaptic protein turnover and now help to approach this question. Interestingly, we found function in a gender-specific manner. In fact, females may be Ambra1+/ − -associated brain enlargement in both genders, thus particularly sensitive to reduced autophagy as suggested also by a uncoupled from the predominantly female behavioral phenotype. recent paper reporting higher basal autophagy activity in the This finding may be important in connection with recent
Translational Psychiatry (2017), 1 – 9 Sexual dimorphism of AMBRA1-associated autistic traits M Mitjans et al 8 suggestions, based on genetically undefined autistic children, 7 Hazlett HC, Gu H, Munsell BC, Kim SH, Styner M, Wolff JJ et al. Early brain develop- where brain volume overgrowth was linked to the emergence and ment in infants at high risk for autism spectrum disorder. Nature 2017; 542:348–351. severity of autistic social deficits, predicting a later autism 8 Piven J, Arndt S, Bailey J, Havercamp S, Andreasen NC, Palmer P. An MRI study of diagnosis based on MRI deep-learning algorithms.7 This obvious brain size in autism. Am J Psychiatry 1995; 152: 1145–1149. discrepancy should stimulate further investigations considering 9 Keller R, Basta R, Salerno L, Elia M. Autism, epilepsy, and synaptopathies: a not rare association. Neurol Sci 2017; 38: 1353–1361. gender, genetics and biological ASD subgroups. 10 Tuchman R, Rapin I. Epilepsy in autism. Lancet Neurol 2002; 1: 352–358. 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Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism. Proc Natl Acad Sci USA 2008; 105: 1710–1715. autistic traits across species. In humans, it will for instance be 18 Schroeder JC, Reim D, Boeckers TM, Schmeisser MJ. Genetic animal models for interesting to systematically screen ASD patients for AMBRA1 autism spectrum disorder. Curr Top Behav Neurosci 2017; 30311–30324. +/ − mutations, particularly female autists. Ambra1 mice may serve 19 Clarke TK, Lupton MK, Fernandez-Pujals AM, Starr J, Davies G, Cox S et al. Com- as a novel multilayered construct-valid genetic model of human mon polygenic risk for autism spectrum disorder (ASD) is associated with cog- autistic phenotypes. nitive ability in the general population. Mol Psychiatry 2016; 21: 419–425. 20 Gaugler T, Klei L, Sanders SJ, Bodea CA, Goldberg AP, Lee AB et al. Most genetic risk for autism resides with common variation. Nat Genet 2014; 46:881–885. CONFLICT OF INTEREST 21 Ehrenreich H, Mitjans M, Van der Auwera S, Centeno TP, Begemann M, Grabe HJ et al. OTTO: a new strategy to extract mental disease-relevant combinations of The authors declare no conflict of interest. GWAS hits from individuals. Mol Psychiatry 2016. 22 Ehrenreich H, Nave KA. Phenotype-based genetic association studies (PGAS)- ACKNOWLEDGMENTS towards understanding the contribution of common genetic variants to schizo- phrenia subphenotypes. Genes 2014; 5:97–105. This work was supported by the Max Planck Society, the Max Planck Förderstiftung, 23 Stepniak B, Kastner A, Poggi G, Mitjans M, Begemann M, Hartmann A et al. the DFG (CNMPB), EXTRABRAIN EU-FP7, the Niedersachsen-Research Network on Accumulated common variants in the broader fragile X gene family modulate Neuroinfectiology (N-RENNT) and EU-AIMS. The research of EU-AIMS receives support autistic phenotypes. EMBO Mol Med 2015; 7: 1565–1579. from the Innovative Medicines Initiative Joint Undertaking under grant agreement n 24 Kastner A, Begemann M, Michel TM, Everts S, Stepniak B, Bach C et al. Autism 115300, resources of which are composed of financial contribution from the beyond diagnostic categories: characterization of autistic phenotypes in schizo- European Union's Seventh Framework Program (FP7/2007–2013), from the EFPIA phrenia. BMC Psychiatry 2015; 15: 115. companies and from Autism Speaks. SHIP is part of the Community Medicine 25 Careaga M, Murai T, Bauman MD. Maternal immune activation and autism Research net of the University Medicine Greifswald, which is funded by the Federal spectrum disorder: from rodents to nonhuman and human primates. Biol Psy- State of Mecklenburg-West Pomerania. Genome-wide data in SHIP have been chiatry 2017; 81:391–401. supported by a joint grant from Siemens Healthineers, Erlangen, and the Federal 26 Ehrenreich H. The impact of environment on abnormal behavior and mental State of Mecklenburg-West Pomerania. We thank all subjects for participating in the disease: to alleviate the prevalence of mental disorders, we need to phenotype study, and all the many colleagues who have contributed over the past decade to the the environment for risk factors. EMBO Rep 2017; 18: 661–665. GRAS data collection. 27 Baron-Cohen S, Cassidy S, Auyeung B, Allison C, Achoukhi M, Robertson S et al. Attenuation of typical sex differences in 800 adults with autism vs. 3,900 controls. PLoS ONE 2014; 9: e102251. AUTHOR CONTRIBUTIONS 28 Halladay AK, Bishop S, Constantino JN, Daniels AM, Koenig K, Palmer K et al. Sex Concept, design and supervision of the study: HE. Data acquisition/analysis/ and gender differences in autism spectrum disorder: summarizing evidence gaps and identifying emerging areas of priority. 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Prediction of complete gene structures in human genomic DNA. article are included in the article’s Creative Commons license, unless indicated J Mol Biol 1997; 268:78–94. otherwise in the credit line; if the material is not included under the Creative Commons 53 Netrakanti PR, Cooper BH, Dere E, Poggi G, Winkler D, Brose N et al. Fast cerebellar license, users will need to obtain permission from the license holder to reproduce the reflex circuitry requires synaptic vesicle priming by munc13-3. Cerebellum 2015; material. To view a copy of this license, visit http://creativecommons.org/licenses/ 14: 264–283. by/4.0/ 54 Ju A, Hammerschmidt K, Tantra M, Krueger D, Brose N, Ehrenreich H. Juvenile manifestation of ultrasound communication deficits in the neuroligin-4 null mutant mouse model of autism. Behav Brain Res 2014; 270:159–164. © The Author(s) 2017
Translational Psychiatry (2017), 1 – 9
8 13th Psychoimmunology Expert Meeting March 3–6, 2016 / Neurology, psychiatry and brain research 22 (2016) 1–25 needed to ameliorate inflammatory pathology caused by bacterial picture anymore, but T regulatory cells are normalized or infections of the CNS. MicroRNA (miR) are short, non-coding RNA increased and inflammatory type T helper subsets (Th1 and that regulate inflammation in bone marrow (BM)-derived myeloid Th17 cells) increased. cells as well as in brain parenchymal cells, e.g. microglia. Among 4. In stages of T regulatory T cell defects, i.e. in early pre-stage these, miR-155 is an important regulator of brain inflammation in bipolar disorder (adolescent children of a bipolar parent) the autoimmune conditions but little is known about its expression expression of inflammatory genes is enhanced in circulating and role during bacterial infection. Experiments presented here monocytes. This is also the case in active disease in later stages analyzed expression and function of miR-155 in the brain during of bipolar disorder. experimental Listeria monocytogenes (Lm) infection of mice. For major depressive disorder (MDD): Infection of C57BL/6 mice with of 2-5LD50 Lm upregulated miR- 5. In all stages of major depressive disorder partial T cell defects 155 measured in whole brain by 24 h following i.p. injection. are observed in FACS analysis, i.e. slightly lower numbers of Similarly, using a model in which lethally infected mice were circulating CD3+ and/or CD4+ T cells. These T cell defects again rescued with antibiotics given 48 h post-infection (PI), miR-155 involve in particular CD4+ CD25+ FoxP3+ T regulatory cells and expression increased at day 3PI, peaked at day 7PI, then declined are most prominent in MDD of over 28 years. slowly despite brain infection being eliminated by day 7 PI. 6. Lymphoid and myeloid growth factors are decreased in the Analysis of mRNA expression in antibiotic treated mice showed serum of MDD cases. maximal expression of Il1b and Tnfa coincided with maximal miR- 7. In MDD patients of <28 years there is a reduced expression of pos 155 expression. In isolated CD11b brain cells, miR-155 immune activation genes in monocytes expression increased at day 7PI but not at day 3 PI suggesting it 8. In late stage MDD (> 28 years) the expression of inflammatory may have a measurable role regulating inflammatory responses in and immune activation genes is enhanced in circulating microglia at that time point. Functional studies measured cytokine monocytes. This enhanced expression correlates to the T À/À expression in brain cells isolated from C57BL/6 and miR-155 regulatory cell defects occurring at that time. A course of 7 mice after overnight culture with heat-killed Lm. Cells from miR- weeks anti-depressive drug treatment increases the number of À/À 155 mice isolated at day 6 & 7PI produced greater concentra- circulating CD4+ CD25+ FoxP3+ T regulatory cells almost two tions of IL-1a, IL-1b, TNF-a, CCL3, and CCL4 than did cells from fold, but does not (yet) have significant effects on inflammatory pos normal mice. Similarly, a higher percentage of TNF-a microglia gene expression in monocytes. was found by intracellular flow cytometry in miR-155À/À mice compared with normal animals. In contrast, cytokine production In summary: and intracellular TNF-a expression in microglia did not differ Major depressive disorder is characterized by defects in lymphoid between genotypes at steady state or day 3PI. These data suggest and myeloid growth factors, relatively low numbers of T regulator miR-155 upregulated in the brain by Lm infection dampens and effector T helper cells, and a reduced monocyte function, apart inflammatory responses in microglia. Thus, enhancing its expres- from a high inflammatory function of monocytes at older age (i.e. sion could be a novel form of adjunctive therapy during bacterial >28 years). meningitis. Bipolar disorder is characterized by increases in lymphoid and myeloid growth factors (compensating initial T cell and myeloid http://dx.doi.org/10.1016/j.npbr.2015.12.018 cell defects?), and in later stages of the disease normal T regulatory cells, relatively high numbers of effector T helper cells and a high inflammatory function of monocytes related to the activity of the Two different immune pathogeneses models for disease. bipolar disorder and major depressive disorder
Hemmo A. Drexhageon behalf of all MOODINFLAM, http://dx.doi.org/10.1016/j.npbr.2015.12.019 PSYCHAID investigators
Erasmus MC, Dr. Molewaterplein 50, Rotterdam, The Netherlands Circulating NMDAR1 autoantibodies of different immunoglobulin classes modulate evolution of Two integrated models 2015 for the immune pathogenesis of lesion size in acute ischemic stroke the two severe mood disorders are proposed. These models are Hannelore Ehrenreich 1,*, Esther Castillo-Gomez 1, bases on the observations of a dynamic course of T cell and Barbara Oliveira 1, Christoph Ott 1, Johann Steiner 2, monocyte abnormalities in patients with either bipolar disorder or Karin Weissenborn 3 major depression as obtained in the MOODINFLAME and 1 PSYCHAID EU projects. The observations are: Clinical Neuroscience, Max Planck Institute of Experimental Medi- For bipolar disorder (BD) cine, and DFG Research Center for Nanoscale Microscopy & Molecular Physiology of the Brain (CNMPB), Go¨ttingen, Germany 2 1. In ‘‘early’’ pre-stages of bipolar disorder (prior to symptoms, i.e. Department of Psychiatry, University of Magdeburg, Magdeburg, in adolescent children of a bipolar parent) partial T cell defects Germany 3 are observed in FACS analysis, i.e. slightly lower numbers of Department of Neurology, Hannover Medical School, Hannover, circulating CD3+ and/or CD4+ T cells. These T cell defects do in Germany particular involve CD4+ CD25+ FoxP3+ T regulatory cells. In *Corresponding author. bipolar twin studies these CD4+ CD25+ FoxP3+ T regulatory cell defects were almost entirely determined by the genetic back Building on the high seroprevalence of autoantibodies (AB) ground of the individual. directed against the N-methyl-D-aspartate-receptor subunit NR1 2. Lymphoid and myeloid growth factors are increased in serum of (NMDAR1) across health and disease, we provide here examples pre-stage bipolar disorder and also in established bipolar where circulating NMDAR1-AB may be of (patho) physiological disorder, such as increases in IL-7, IGF-BP2, sCD25 and SCF. relevance. Dependent on acute or chronic dysfunction of the blood- 3. In established bipolar disorder (and also in older children of a brain-barrier (BBB), they may play the role of a ‘double-edged bipolar parent) defects in T regulatory cells do not determine the sword’. We further report that NMDAR1-AB not only bind to 13th Psychoimmunology Expert Meeting March 3–6, 2016 / Neurology, psychiatry and brain research 22 (2016) 1–25 9 neurons, but also to other cell types in the brain, and that their express NMDAR1 which may influence their neuron/axon support- immunoglobulin class does not seem to affect their function in an ing, detoxifying, metabolic and protection/defense properties. internalization assay using human cortical neurons or in modu- Blocking these NMDAR1 functions by AB might variably contribute lating stroke outcome of human patients. to the modulation of brain functions in these conditions and We previously reported high seroprevalence (age-dependent ultimately co-determine stroke outcome. up to >20%) of N-methyl-D-aspartate-receptor subunit NR1 To substantiate these considerations, we provide here a first (NMDAR1) autoantibodies (AB) in healthy and neuropsychiatrical- composite figure of our work in progress which illustrates scattered ly ill subjects (N = 4236) (Dahm et al., 2014). Neuropsychiatric binding of a human NMDAR1-AB (IgG; 1:1000) positive serum of a syndrome relevance was restricted to individuals with compro- stroke patient to endothelial cells (CD13), NG2 cells and astrocytes mised blood-brain-barrier (BBB), e.g. APOE4 carrier status, both (GFAP), but not to microglia (Iba1) in healthy mouse hippocampus. clinically and experimentally (Hammer et al., 2013; Hammer et al., Expectedly, also neurons are specifically labeled by this human AB 2014). Considering earlier data in rats (During et al., 2000), and the (data not shown). In conditions of hypoxia/ischemia, inflamma- fact that excessive NMDAR-stimulation is regarded as a lead tion, demyelination or degeneration, but also during normal pre- mechanism mediating stroke-associated brain damage, we hy- and postnatal development, aging, and perhaps even during pothesized that NMDAR1-AB may modulate stroke outcome. We learning processes, binding of NMDAR1-AB to the different cell further hypothesized that in ischemic stroke patients the Ig class of types may be increased or altered (Fig. 1). serum NMDAR1-AB would not matter regarding their effects on In conclusion, much more work will have to go into stroke outcome. This latter hypothesis was based on our previous understanding consequences of circulating NMDAR1-AB in disease studies, using IgG, IgA and IgM NMDAR1-AB derived from human states with accompanying BBB breakdown, where these AB may serum in the ApoEÀ/À mouse model of BBB dysfunction with possibly play the role of a double-edged sword. Reduction of lesion comparable behavioral consequences (Hammer et al., 2014). size during acute ischemia may be followed by an increased risk of Patients with acute ischemic stroke in the middle cerebral cognitive decline, epilepsy or psychosis upon extended AB artery territory (N = 464) were prospectively enrolled for a exposure of the brain, due to a lastingly compromised BBB after biomarker study with treatment as usual. Blood for NMDAR1-AB stroke (5). Further studies will be necessary to evaluate potential measurements was collected within 3-5 hours after stroke. APOE4 benefits of passive (rather than active) immunization under carrier status was determined as indicator of a pre-existing leaky carefully controlled conditions. BBB. Evolution of lesion size (delta day7-1) in diffusion weighted magnetic resonance imaging (MRI) was primary outcome param- eter. In subgroups, NMDAR1-AB measurements were repeated on days 2 and 7 (Zerche, Weissenborn, & Ott, 2015). For further mechanistic insight, wildtype and transgenic mouse brain sections, ApoEÀ/À mice and human IPS-derived cortical neurons were employed. Of 464 patients (mean age 68 years), 21.6% were NMDAR1-AB positive (IgM, A, or G), 21% were APOE4 carriers. Patients with MRI data available on days1 and 7 were divided into 4 groups: (1) ABÀ/ APOE4À (N = 236); (2) AB+/APOE4À (N = 64); (3) ABÀ/APOE4+ (N = 64) and (4) AB+/APOE4+ (N = 20). Groups were comparable in stroke-relevant presenting characteristics. The AB+/APOE4À group had a smaller mean delta lesion size, compared to the ABÀ/ APOE4À group, suggesting a protective effect of circulating NMDAR1-AB. Conversely, the AB+/APOE4+ group had the largest mean delta lesion area, indicating a damaging influence of NMDAR1-AB in APOE4 carriers. Hence, dependent on BBB integrity before an acute ischemic stroke, pre-existing NMDAR1-AB appear to be beneficial or detrimental. NMDAR1-AB effects on outcome were comparable in carriers of all 3 Ig classes (Zerche et al., 2015). NMDAR1-AB titers in serum dropped on day2 and remounted by day7 after stroke (Zerche et al., 2015), consistent with the brain acting as immunoprecipitating trap for brain antigen-directed AB in conditions of BBB breakdown, later followed by boosting of AB production by plasma cells. This boosting likely occurs as a consequence of the abrupt exposure of the immune system to brain antigens upon major BBB disruption. The serum titer drop observed after stroke was reproducible in the ApoEÀ/À mouse model of BBB disturbance (still unpublished). Since in the stroke study we had essentially comparable results for carriers of IgM, IgA and IgG (5), we employed human IPS-derived cortical neurons to Fig. 1. Representative tilescan confocal images showing a panoramic view of the compare Ig class effects in vitro using a receptor internalization hippocampus of 4-week old mice, immunostained with dialyzed serum of a stroke patient, seropositive (IgG) for NMDAR1-AB (secondary AB: goat anti-human IgG- assay (3). We found comparable internalization of NMDAR1 by all Cy3 = red, Jackson Immuno Research; A–D). Nuclei in all images are stained with NMDAR1-AB isotypes (still unpublished). DAPI (blue). Endothelial cells (green; A), astrocytes (green, B) and microglia (green; Many NMDAR1 expressing cell types in the brain apart from C) are stained with commercial primary AB [rat anti-CD13, BD Biosciences; mouse neurons likely contribute to short-term and/or long-term outcome anti-GFAP, Boehringer Mannheim; rabbit anti-Iba1, WaKo], followed by the respective secondary AB, in a wild-type mouse hippocampus. D: NG2-GFP positive after stroke and may be functionally modulated by circulating cells (green) in the brain of an NG2-CreERT2 transgenic mouse (tamoxifen NMDAR1-AB that suddenly gain access to the brain in larger injections at postnatal days 26 and 27). Scale bar: 250 mm (left panel), 25 mm amounts. In fact, endothelial cells, oligodendrocytes and astrocytes (middle panel), 65 mm (right panel). 10 13th Psychoimmunology Expert Meeting March 3–6, 2016 / Neurology, psychiatry and brain research 22 (2016) 1–25
References significant changes in M1 and M2 associated surface markers which indicate modified activation patterns of microglia Dahm, L., Ott, C., Steiner, J., Stepniak, B., Teegen, B., Saschenbrecker, S., et al. (2014). Seroprevalence of autoantibodies against brain antigens in health and populations based on prenatal infection. The shift towards a disease. Annals of Neurology, 76(1), 82–94. M1 phenotype of the microglial cells of Poly(I:C) descendants During, M. J., Symes, C. W., Lawlor, P. A., Lin, J., Dunning, J., Fitzsimons, H. L., et al. points to the significance of immunological processes in (2000). An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy. Science, 287, 1453–1460. schizophrenia. Hammer, C., Stepniak, B., Schneider, A., Papiol, S., Tantra, M., Begemann, M., et al. (2013). Neuropsychiatric disease relevance of circulating anti-NMDA receptor http://dx.doi.org/10.1016/j.npbr.2015.12.021 autoantibodies depends on blood-brain barrier integrity. Molecular Psychiatry, 19(10), 1143–1149. Hammer, C., Zerche, M., Schneider, A., Begemann, M., Nave, K. A., & Ehrenreich, H. (2014). Apolipoprotein E4 carrier status plus circulating anti-NMDAR1 Anti-inflammatory characteristics of autoantibodies: association with schizoaffective disorder. Molecular Psychiatry, acetylcholine esterase inhibitor Physostigmine 19(10), 1054–1056. Zerche, M., Weissenborn, K., Ott, C., Dere, E., Asif, A. R., Worthmann, H., et al. (2015). and its potential role in preventing Preexisting serum autoantibodies against the NMDAR subunit NR1 modulate postoperative cognitive dysfunction evolution of lesion size in acute ischemic stroke. Stroke, 46(5), 1180–1186. Lea Fritzenschaft *, Simon Zolg, Benedikt Zujalovic, http://dx.doi.org/10.1016/j.npbr.2015.12.020 E. Marion Schneider, Eberhard Barth Department Anesthesiology, University Hospital Ulm, Albert-Einstein- Prenatal immune challenge induces changes of Allee 23, Ulm 89081, Germany microglial surface markers in an animal model *Corresponding author. of schizophrenia Manuela Eßlinger 1,*, Simone Wachholz 1, Postoperative cognitive dysfunction (POCD) is a complication Marie-Pierre Manitz 1, Rainer Sommer 1, affecting cognitive and locomotor functions in patients suffering Jennifer Plu¨ mper 1, Awatef Esshili 1, from brain trauma as well as severe sepsis and intensive care Alexandra Gerhard 1,2, Georg Juckel 1,2, treatment. Symptoms include clouding of consciousness and Astrid Friebe 1,2 memory loss as well as impairment of attention, language and locomotor abilities. 1 Workgroup of Psychoneuroimmunology, Department of Psychiatry, Various possible pathologic causes for POCD have been proposed, ZKF1 2.052 Ruhr University Bochum, Universita¨tsstr. 150, 44801 but the most popular hypothesis is a central cholinergic deficiency Bochum, Germany caused by dysregulation of cholinergic anti-inflammatory pathways, 2 LWL University Hospital, Alexandrinenstr. 1-3, 44791 Bochum, leading to increased and chronic inflammation. Acetylcholine, the Germany main mediator of the cholinergic anti-inflammatory pathway, *Corresponding author. suppresses NF-kB activation and inhibits the release of pro- inflammatory cytokines (e.g. tumor necrosis factor, interleukin Epidemiological studies indicate that maternal infection (IL)-1b, IL-6, and IL-18) by macrophages. The anti-inflammatory during pregnancy is associated with a higher incidence of properties of acetylcholine are mediated through the nicotinic schizophrenia in the offspring. It is hypothesized that an acetylcholine receptors, especially the alpha-7 subunit is crucial for inflammatory immune response interferes with normal fetal the anti-inflammatory effect. Available acetylcholine is degraded by brain development. Long-lasting changes of this developmental acetylcholinesterase. As pretested with healthy donors, we are going disruption might provide a neural basis for enhanced vulnerabili- establish whole blood assays to determine the acetylcholine and ty which might manifest in schizophrenia in the adult descen- buturylesterase activities from of patients treated by intensive care dants. We suggest microglia as possible initiator for this enhanced and a high risk to manifest POCD. So far, ex vivo whole blood vulnerability. In previous studies we showed a significant stimulation assays have were applied to test whether inflammation alteration of microglial cell numbers in the adolescent offspring and or/enzyme activities can be modulated by exogenous Physostig- of Poly(I:C) injected mice compared to controls. Here we suggest a mine. long term effect on microglia properties manifested in an altered Physostigmine is a reversible acetylcholinesterase inhibitor and expression pattern of pro- and anti-inflammatory surface markers therefore a cholinergic stimulant. due to prenatal immune challenge. We hypothesize that Physostigmine can activate the choliner- We used the Poly(I:C) mouse model of maternal immune gic anti-inflammatory pathway and prevent the activation of activation to analyze pro- and anti-inflammatory surface macrophages via stimulation of the alpha-subunit of the nicotinic markers on microglia of descendants from Poly(I:C) vs. saline acetylcholine receptor. exposed BALB/c mice at postnatal days 30 (puberty) and 100 Blood samples from volunteer donors and from ICU patients (adulthood) by flow cytometric analysis. Analysis showed clear will be collected into the TruCulture whole blood culture system. sex dependent differences in microglia surface marker expres- As a stimulant, LPS will be added to mimic postoperative infection. sion between treatment groups. At day 30, female offspring from The anti-inflammatory properties of Physostigmine will be Poly(I:C) exposed mice showed significant microglia activation determined by flow cytometry and cytokine quantification using by up-regulation of M1 related activation markers and down- the. Immulite 1000 ELISA. regulation of M2 associated markers. Microglia activation We expect that the application of Physostigmine significantly was not detected in adult female mice. In contrast, male reduces the amount of activated macrophages and cytokines. Our descendants did not show signs of activation neither in puberty results will contribute to our understanding of the complex nor in adulthood. The alteration of microglia surface marker molecular pathology in POCD. expression corresponds with our prior study, in which we showed an increased microglial cell number in the brains of the adolescent offspring from Poly(I:C) exposed mice. Here we show http://dx.doi.org/10.1016/j.npbr.2015.12.022
Widespread Expression of Erythropoietin Receptor in Brain and Its Induction by Injury
Christoph Ott,1 Henrik Martens,2 Imam Hassouna,1,3 Bárbara Oliveira,1 Christian Erck,2 Maria-Patapia Zafeiriou,4 Ulla-Kaisa Peteri,1 Dörte Hesse,5 Simone Gerhart,1 Bekir Altas,6 Tekla Kolbow,2 Herbert Stadler,2 Hiroshi Kawabe,6 Wolfram-Hubertus Zimmermann,4 Klaus-Armin Nave,7,8 Walter Schulz-Schaeffer,9 Olaf Jahn,5,8 and Hannelore Ehrenreich1,8
1Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany; 2Synaptic Systems GmbH, Göttingen, Germany; 3Physiology Unit, Zoology Department, Faculty of Science, Menoufia University, Egypt; 4Institute of Pharmacology, University Medicine Göttingen, Göttingen, Germany; 5Proteomics Group, 6Molecular Neurobiology, and 7Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen, Germany; 8DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany; and 9Department of Neuropathology, University Medicine Göttingen, Göttingen, Germany
Erythropoietin (EPO) exerts potent neuroprotective, neuroregenerative and procognitive functions. However, unequivocal dem- onstration of erythropoietin receptor (EPOR) expression in brain cells has remained difficult since previously available anti-EPOR antibodies (EPOR-AB) were unspecific. We report here a new, highly specific, polyclonal rabbit EPOR-AB directed against different epitopes in the cytoplasmic tail of human and murine EPOR and its characterization by mass spectrometric analysis of immuno- precipitated endogenous EPOR, Western blotting, immunostaining and flow cytometry. Among others, we applied genetic strat- egies including overexpression, Lentivirus-mediated conditional knockout of EpoR and tagged proteins, both on cultured cells and tissue sections, as well as intracortical implantation of EPOR-transduced cells to verify specificity. We show examples of EPOR expression in neurons, oligodendroglia, astrocytes and microglia. Employing this new EPOR-AB with double-labeling strategies, we demonstrate membrane expression of EPOR as well as its localization in intracellular compartments such as the Golgi apparatus. Moreover, we show injury-induced expression of EPOR. In mice, a stereotactically applied stab wound to the motor cortex leads to distinct EpoR expression by reactive GFAP-expressing cells in the lesion vicinity. In a patient suffering from epilepsy, neurons and oligodendrocytes of the hippocampus strongly express EPOR. To conclude, this new analytical tool will allow neuroscientists to pin- point EPOR expression in cells of the nervous system and to better understand its role in healthy conditions, including brain de- velopment, as well as under pathological circumstances, such as upregulation upon distress and injury. Online address: http://www.molmed.org doi: 10.2119/molmed.2015.00192
INTRODUCTION tive effects make EPO an attractive can- cific cytokine-type-1 transmembrane re- The growth factor erythropoietin didate for treating human brain disease ceptor molecules, each with a calculated (EPO) was named based on its first dis- (6–7), and an important target of neuro- molecular mass of 59 kDa, that together covered effects on cells of the hematopoi- science research. In 1989, the EPO recep- form the classical homodimeric EPOR etic system. For >20 years it has been tor (EpoR) was first cloned in mice (8), (2,11). Binding of EPO to its receptor in- shown to act on other tissues, including soon followed by cloning and characteri- duces a conformational change, initiating the brain (1–5). Its remarkable neuropro- zation of the human EPOR gene (9–10). EPOR-associated JAK2 transphosphory- tective, neuroregenerative and procogni- A single EPO molecule binds to two spe- lation and multiple, cell-type-specific, downstream signal transduction cas- cades. These cascades include signal transducers and activators of transcrip- Address correspondence to Hannelore Ehrenreich, Clinical Neuroscience, Max Planck In- tion (STATs), phosphatidylinositol-3 ki- stitute of Experimental Medicine, Göttingen, Germany. Phone: +49-551-3899615; Fax: +49- nase (PI3K)/AKT, RAS/extracellular 551-3899670; E-mail: [email protected]. signal-regulated kinase (ERK1/2), nu- Submitted August 25, 2015; Accepted for publication September 1, 2015; Published Online clear factor κB (NF-κB). Activation of (www.molmed.org) September 1, 2015. these signaling cascades leads to further activation of antiapoptotic factors and pathways, stimulation of cell differentia- tion, including induction of cellular
MOL MED 21:803-815, 2015 | OTT ET AL. | 803 EPOR IN BRAIN
shape-change and growth, or modula- cluded. Recognizing this critical issue in tion and the immunoglobulin subclass tion of plasticity, in a cell-type and stim- EPO/EPOR research, we aimed at gener- was determined (IgG2b). The AB is ulation-dependent manner (5,12–13). ating reliable EPOR-AB. We present here called “ntEPOR-AB” in this article. Antibodies against EPOR (EPOR-AB) the comprehensive characterization of a have been widely used to characterize novel, highly specific EPOR-AB, using an Cell Culture EPOR expression and localization, but array of state-of-the-art technologies. This Cell lines. The following human cell cell surface EPOR expression is low, even new AB tool may help overcome the de- lines were used: The EPO-dependent in stimulated states, and, most impor- scribed obstacles and lead to revisiting megakaryoblastic leukemia UT-7 cell tantly, all commercially available some of the reported data. line, the erythroleukemia cell line EPOR-AB have been hampered by non- OCIM-1, the mouse microglia cell line specific cross-reactivities, calling into MATERIALS AND METHODS EOC-20 and HEK293 FT cells. The EPO- question the literature based exclusively dependent megakaryoblastic leukemia on them. This, in turn, raised discussions Generation of EPOR-AB UT-7 cell line was from Drorit Neumann within the scientific community, ques- Polyclonal AB. Two rabbits were im- of Tel Aviv University in Israel. This cell tioning the expression of EPOR in extra- munized with a purified recombinant line was cultured in IMDM with 1% hematopoietic tissues (14–16). These dis- protein corresponding to amino acids GlutaMAX supplement (Invitrogen, cussions were likely nurtured by (AA) 273-508 (intracellular C-terminus) Darmstadt, Germany), 10% FBS, conflicts of interest, trying to restrict the of the unprocessed human EPOR. The 100 U/mL penicillin and 100 μg/mL effects of EPO, a highly attractive com- coding sequence was generated by gene streptomycin (all Life Technologies pound commercially for the anemia mar- synthesis (Geneart, Regensburg, Ger- GmbH, Darmstadt, Germany) and ket, to hematopoiesis. Nevertheless, they many) and ligated via EcoRI and HindIII 2 IU/mL EPO (NeoRecormon, Roche, made it very obvious that the existing into the bacterial expression vector Welwyn Garden City, UK). The erythro - EPOR-AB were essentially unreliable, pASK-IBA37+ (IBA-Lifescience, Göttingen, leukemia cell line OCIM-1 (DSMZ and that the production and thorough Germany). The recombinant His-Tag fu- GmbH, Braunschweig, Germany) was characterization of new and more spe- sion protein was expressed and purified cultured in IMDM with 1% GlutaMAX cific EPOR-AB had to be seen as a major by Ni-NTA affinity chromatography ac- supplement, 10% FBS and 100 U/mL challenge for the future (14,17–18). cording to the manufacturer’s manual. penicillin and 100 μg/mL streptomycin Independent of work based on EPOR- Crude antiserum SA7378 was affinity pu- (all Life Technologies GmbH). AB, genetically altered mice helped to rified with the immunogen coupled to The mouse microglia cell line EOC-20 demonstrate that EPOR signaling is nec- CNBr Sepharose (GE-Healthcare, (ATTC LGC Standards, Wesel, Germany) essary for normal brain development (19) Freiburg, Germany). The AB is called was cultured in DMEM with 1 mmol/L and that it has a distinct function in neu- “ctEPOR-AB” in this article. sodium pyruvate, 0.15%sodium bicar- rogenesis (20). In addition, EPO and Monoclonal AB. AB producing hy- bonate, 10% FBS, 100 U/mL penicillin, EPOR mRNA are expressed in brain tis- bridomas were generated by Synaptic 100 μg/mL streptomycin (all Life Tech- sue (21), and specific binding sites for Systems (Göttingen, Germany; see also nologies GmbH) and 0.01 μg/mL murine EPO in the brain have been demonstrated http://sysy.com/services/index.php) M-CSF (PAN-Biotech, Aidenbach, Ger- in mouse and humans by means of radio- as follows: Three 8–10 wks old BALB/c many). HEK293 FT cells (Sigma-Aldrich, labeled EPO (22–23). In cell culture, female mice were subcutaneously immu- Taufkirchen, Germany) were cultured in mRNA expression combined with func- nized with a synthetic peptide corre- DMEM with 5% FBS and 100 U/mL pen- tional assays, for example, altered phos- sponding to AA 25-39 (extracellular icillin and 100 μg/mL streptomycin (all phorylation of second messenger path- N-terminus) of unprocessed human Life Technologies GmbH). ways induced by EPO in microglia, EPOR precursor coupled to KLH via a Human IPS cells. Human material was served to prove specific EPOR expression C-terminal cysteine over a period of 75 d. used in accordance with ethical guidelines in the absence of reliable EPOR-AB (24). Cells from the knee lymph nodes were and the Helsinki Declaration. Subjects The fact that cellular EPOR protein ex- fused with the mouse myeloma cell line gave informed consent regarding genera- pression has been difficult to assess P3X63Ag8.653 (ATCC CRL-1580). Result- tion and use of IPS cells or scientific in- strongly limited the in-depth investiga- ing hybridomas were screened by direct vestigation of brain samples. Human fi- tion of the EPO/EPOR system. Particu- enzyme-linked immunosorbent assay broblasts were reprogrammed using a larly in the human brain, the study of its (ELISA) against the immunogen and im- nonintegrative RNA-based virus to in- (patho-) physiological role has been munofluorescence on 3T3 NIH fibrob- duce the expression of four reprogram- highly constrained since additional means lasts overexpressing full-length human ming factors: OCT4, SOX2, KLF4 and c- of verification as used in experimental an- EPOR. Clone 45A3, used in this study, MYC (CytoTune-iPS 2.0 Sendai imals and cell cultures are naturally ex- was recloned two times by limiting dilu- Reprogramming Kit, Life Technologies
804 | OTT ET AL. | MOL MED 21:803-815, 2015 RESEARCH ARTICLE
GmbH). After transduction, IPS cells available upon request. Primary mouse main, ~40 kDa) and an anchored human (clones isAu1-3; isAu3-2) were adapted to astrocytes prepared from P0-2 forebrains EPOR (lacks the N-terminus and the C- a feeder-free culture system (Matrigel ma- of EpoR-fl/fl mice were used. The prepa- terminus, ~12 kDa). trix, Corning, Wiesbaden, Germany) and ration and culture conditions of these cultured in TeSR-E8 medium (STEMCELL cells are described in detail elsewhere STAT5 Phosphorylation Assay Technologies SARL, Cologne, Germany). (26). The cells were infected with This assay is described in detail else- Primary mouse cell culture. The prepa- lentiviruses at d 1 in vitro. The viral con- where (17). In brief, UT-7 or OCIM-1 cells ration and culture conditions of primary structs contained either a cassette for were serum- and EPO-deprived overnight mouse oligodendrocytes and microglia GFP-only (control) or a cassette for GFP (1% FBS in IMDM, both Life Technologies are described in detail elsewhere (24–25). and Cre-recombinase. Protein was ex- GmbH). On the next day, they were incu- In brief, oligodendrocytes were prepared tracted on d 10 in vitro. bated with different concentrations of from the forebrains of newborn P1-2 recombinant human EPO (rhEPO, NMRI mice. After differentiation, oligo- EOC-20 Cell Transduction NeoRecormon, Roche) or control solution dendrocyte precursors were shaken off For viral transduction, 100,000 EOC-20 for 15 min, followed by protein extraction from a bottom layer of astrocytes and cells were seeded in 12-well plates for Western blotting. Immunodetection seeded in Super-Sato medium (DMEM overnight. The next day, the medium was done with antiphosphorylated STAT5 with high glucose supplemented with was partially exchanged by DMEM (Life (1:500, Cell Signaling, Danvers, MA, USA) B-27 supplement, 2 mmol/L GlutaMAX, Technologies GmbH) containing viral su- and GAPDH (1:5000, Enzo Life Sciences, 1 mmol/L sodium pyruvate, 1% horse pernatant and 8 μg/mL polybrene Farmingdale, NY, USA); 20 μg of protein serum [HS], 50 U/mL penicillin and (Sigma-Aldrich). The ecotropic virus par- was loaded for SDS-PAGE. 50 μg/mL streptomycin [all from Life ticles used were derived from a pMOWS Technologies GmbH] and 0.5 mmol/L tri- vector encoding N-terminally HA-tagged MAPK Phosphorylation Assay iodothyronine, and 0.52 mmol/L full-length human EPOR and Transduced EOC-20 cells were kept in L-thyroxine [both Merck, Darmstadt, Ger- puromycin-resistant cassette (gift from serum-free DMEM (Life Technologies many]). For primary microglia, newborn Ursula Klingmüller, DKFZ Heidelberg, GmbH) overnight. Then, cells were incu- C57BL6 mice (P0-P1) were used. The cell Germany) (27). Next, the 12-well plates bated with different concentrations of suspension derived from their forebrains were centrifuged for 3 h at 330g at room rhEPO (NeoRecormon, Roche) or the re- was seeded in high glucose DMEM me- temperature. The medium was ex- spective control solution for 10 min, fol- dium with 10% HS, 1% GlutaMAX supple- changed again to DMEM with 1 mmol/L lowed by protein extraction for Western ment, 50 U/mL penicillin and 50 μg/mL sodium pyruvate, 0.15% sodium bicar- blotting. Immunodetection was done streptomycin (all from Life Technologies bonate, 10% FBS, 100 U/mL penicillin, with antiphosphorylated MAPK (1:1000), GmbH). Half of the microglia-conditioned 100 μg/mL streptomycin (all Life Tech- anti-MAPK (1:5000) and anti-α-tubulin medium was exchanged by fresh medium nologies GmbH) and 0.01 μg/mL murine (1:5000, all Sigma-Aldrich); 15 μg of pro- 3–4 d later, and at d 7 the medium was M-CSF (PAN-Biotech). The medium was tein was loaded for SDS-PAGE. partially replaced by L929-conditioned supplemented 1 d later with 6 μg/mL medium. Primary microglia were de- puromycin (Sigma-Aldrich) for selection Animal Experiments tached by shaking of flasks and seeded in of successfully transduced EOC-20 cells. All experiments were approved by and serum-free microglial growth medium After successful transduction, cells were conducted in accordance with the regula- (high glucose DMEM with 1 mmol/L constantly cultured in the presence of tions of the local Animal Care and Use sodium pyruvate, 1.5 g/L sodium bicar- 6 μg/mL puromycin. Committee (Niedersächsisches Lan- bonate, 100 U/mL penicillin and desamt für Verbraucherschutz und 100 μg/mL streptomycin [all from Life HEK293 FT Cell Transfection Lebensmittelsicherheit [LAVES]). Technologies GmbH]). HEK293 FT cells were transfected with Stereotactic cell implantation. Male Lipofectamine 2000 reagent (Life Tech- C57BL/6N mice, 8 wks old, were used. Lentiviral Transduction of Primary nologies GmbH) according to the Animals were injected intraperitoneally Cells manufacturer’s instructions. The (i.p.) with carprofen (5 mg/kg Rimadyl, EpoR conditional mouse mutants with pEuExpress-hEPOR vector (Synaptic Sys- Pfizer, Berlin, Germany) 2 h before sur- floxed exons 3-6 were generated on tems, Göttingen, Germany) was used to gery. Under anesthesia (0.276 mg/g tri- the C57BL/6 background by standard transfect the cells with full-length human bromoethanol, Sigma-Aldrich, St. Louis, procedures using mutant ES cells EPOR (~60 kDa), full-length murine MO, USA), mice were positioned in a (EPD0316_5_A03) from the International EpoR (~60 kDa), an N-terminally HA- stereotactic frame and a small, midline Mouse Phenotyping Consortium. Details tagged and C-terminally truncated scalp incision was made. A hole was will be published elsewhere and are human EPOR (lacks the intracellular do- drilled over the left cranial hemisphere at
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a position 1.5 mm anterior and 1.0 mm lysate were incubated for 2 h at 4°C. Af- spectrometric protein identification were lateral to the bregma. Using a sterile terward, beads were centrifuged and performed in parallel on precast Nu- 10-mL Hamilton syringe with a 26-gauge washed. For immunoblot analysis, EPOR PAGE 4% to 12% Bis-Tris gradient gels needle, 15,000 transduced EOC-20 cells was eluted from the beads by repeated using a 3-(N-morpholino)propanesulfonic (description see above) or medium only boiling in Laemmli buffer at 95°C. For acid (MOPS) buffer system according to (DMEM without phenol-red, Life Tech- mass spectrometric protein identification, the manufacturer (Invitrogen). As a pro- nologies GmbH) were slowly (over EPOR was eluted as before but with a tein ladder we used SeeBlue Plus2 2 min) implanted 2 mm deep into the left nonreducing SDS-buffer (without prestained in this gel system (Life Tech- M2 motor cortex. After implantation, the β-mercaptoethanol) to prevent masking of nologies GmbH). Proteins were either vi- needle was left in place for 2 min, then the EPOR by excess AB heavy chains in sualized by colloidal Coomassie staining slowly withdrawn from the brain and the subsequent gel electrophoresis. To in- (gel 1) or transferred on PVDF mem- the skin incision closed with sterile su- crease the efficiency of EPOR capture branes and immunodetected as described ture. Directly after the skin incision was from UT-7 protein lysates, two consecu- above (gel 2). closed, the animals received carprofen tive IPs with fresh beads were performed Protein identification. Gel regions of i.p. for pain treatment, which was re- in a way that the flow-through of the first interest were identified by overlaying peated every 6-8 h (5 mg/kg Rimadyl, IP was used as input for the second. images from colloidal Coomassie stain- Pfizer). At 24 h after surgery, animals Eluted proteins were precipitated by ing and immunodetection in the Delta were anesthetized i.p. (0.276 mg/g tribro- methanol/chloroform treatment (30). Pel- 2D image analysis software (Decodon, moethanol, Sigma-Aldrich) and perfused lets were solubilized in reducing sample Greifswald, Germany). Gel bands were transcardially with 0.9% saline followed buffer and pooled prior to electrophoresis. excised manually and subjected to auto- by 4% formaldehyde in PBS. As starting material for mass spectrome- mated in-gel digestion with trypsin as Labeling of oligodendrocyte precursor try, 4 mg protein lysate from UT-7 cells described previously (31). Tryptic pep- cells in vivo. For induction of CreERT2- was used. tides were dried down in a vacuum cen- activity in NG2-Cre-ERT2:R26R-td- SDS-PAGE and Western blots. SDS- trifuge, redissolved 0.1% trifluoro-acetic tomato-mEGFP mice (28–29), 100 mg/kg PAGE was performed with self-made acid and spiked with 2.5 fmol/μL of tamoxifen (dissolved in corn oil; Sigma- 10% SDS-polyacrylamide gels. As a pro- yeast enolase1 tryptic digest standard Aldrich, Taufkirchen, Germany) was in- tein ladder we used PageRuler Plus (Waters Corporation, Milford, MA, USA) jected i.p. at postnatal d 26 and 27. Ani- prestained and SeeBlue Plus2 prestained for quantification purposes (32). mals were anesthetized i.p. (0.276 mg/g in this gel system (both Life Technologies Nanoscale reversed-phase UPLC separa- tribromoethanol, Sigma-Aldrich) 72 h GmbH). For all Western blotting, the fol- tion of tryptic peptides was performed later and perfused transcardially with lowing protein amounts were loaded: with a nanoAcquity UPLC system 0.9% saline followed by 4% formaldehyde 15 μg for lysates derived from cell lines; equipped with a Symmetry C18 trap col- in PBS. 20 μg for lysates derived from primary umn (5 μm, 180 μm × 20 mm) and a BEH cultures; 50 μg for lysates derived from C18 analytical column (1.7 μm, 75 μm × Detection of EPOR tissue. Afterward, proteins were trans- 100 mm) (Waters Corporation). Peptides Immunoprecipitation (IP). UT-7 protein ferred to a nitrocellulose membrane and were separated over 60 min at a flow lysates were obtained using an IP buffer blocked with 4% milk powder and 4% rate of 300 nL/min with a linear gradient (150 mmol/L NaCl, 20 mmol/L Tris, 1 HS in Tris buffered saline with 0.05% of 1% to 45% mobile phase B (acetonitrile mmol/L EDTA, 10% glycerol, pH = 7.4) Tween 20. Membranes were incubated containing 0.1% formic acid) while mo- containing 1% Triton X-100. Before IP, with ctEPOR-AB (1:2000, Synaptic Sys- bile phase A was water containing 0.1% lysates were diluted 1:1 with IP buffer to tems) at 4°C overnight. For all EPOR im- formic acid. Mass spectrometric analysis obtain 0.5% Triton X-100. For the EPOR IP, munoblots, membranes were washed, of tryptic peptides was performed using protein-G sepharose beads were cova- blocked again and incubated with don- a Synapt G2-S quadrupole time-of-flight lently linked to ctEPOR-AB (Synaptic Sys- key anti-rabbit IRDye 800 AB (1:10000, mass spectrometer equipped with ion tems) with 40 mmol/L dimethyl-pimelim- Rockland, Limerick, PA, USA) for 1 h at mobility option (Waters Corporation). idate (Sigma-Aldrich). Bead slurry room temperature. Primary mouse AB Positive ions in the mass range m/z 50 to (200 μL; Thermo Scientific, Waltham, MA, were detected with donkey anti-mouse 2000 were acquired with a typical resolu- USA) was cross-linked with 400 μg IRDye 800 AB (1:10000, Rockland) for 1 h tion of at least 20,000 FWHM (full width ctEPOR-AB or 400 μg of the IgG fraction at room temperature. After washing, the at half maximum) and data were lock from the same rabbit before immuniza- membranes were scanned with Odyssey mass corrected after acquisition. With the tion. For EPOR IP from UT-7 protein imager (LI-COR Biosciences, Lincoln, NE, aim of increasing the sequence coverage lysates, 9 μg of ctEPOR-AB coupled to USA) and analyzed with the Image Stu- of the identified proteins, analyses were protein-G sepharose per 1 mg protein dio software. Gel electrophoresis for mass performed in the ion mobility-enhanced
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data-independent acquisition mode (1:100, BD Biosciences, Heidelberg, Ger- X-100 in PBS for 1.5 h at room tempera- (33–34) with drift time–specific collision many), rat anti-NG2 (1:250, gift from ture: donkey anti-rabbit Alexa Fluor 594, energies (35). For protein identification, Jacqueline Trotter, University of Mainz, donkey anti-mouse Alexa Fluor 488 (both continuum LC-MS data were processed Germany), goat anti-human Oct-3/4 (1:40, 1:500; Life Technologies GmbH), donkey and searched using Waters ProteinLynx R&D Systems, Minneapolis, MN, USA). anti-chicken Alexa Fluor 488 (1:250), goat Global Server version 3.0.2 (36). A cus- After washing, cells were incubated with anti-guinea pig Cy5 (1:300, both Jackson tom database was compiled by adding the following secondary AB in 0.2% Triton ImmunoResearch). Cell nuclei were visu- the sequence information for yeast eno- X-100 with 1% NHS in PBS for 1 h at alized with DAPI dissolved in H2O lase 1 and porcine trypsin to the room temperature: donkey anti-rabbit (0.01 μg/mL, Sigma-Aldrich). After wash- UniProtKB/Swiss-Prot human proteome Alexa Fluor 488 and anti-goat Alexa Fluor ing in PBS, sections were mounted on (UniProtKB release 2015_06, 20,206 en- 488, donkey anti-mouse Alexa Fluor 568, Super Frost microscopic slides, dried and tries) and by appending the reversed se- donkey anti-rabbit Alexa Fluor 594 (all covered with Aqua-Poly/Mount (Poly- quence of each entry to enable the deter- 1:500, Life Technologies GmbH) and goat sciences). All stainings were scanned by mination of false discovery rate (FDR). anti-rat Alexa Fluor 488 (1:250, Jackson confocal microscopy (TCS SP5-II, Leica). Precursor and fragment ion mass toler- ImmunoResearch, West Grove, PA, USA). Illustration was done using Imaris 7.5.1 ances were automatically determined by Primary microglia were counterstained (www.bitplane.com [Bitplane AG, Zurich, PLGS 3.0.2 and were typically below with tomato lectin Alexa Fluor 488 (1:250, Switzerland]). 5 ppm for precursor ions and below Vector Laboratories, Burlingame, CA, Immunohistochemistry on paraffin- 10 ppm (root mean square) for fragment USA). Cell nuclei were visualized with embedded human brain sections. Brain μ μ ions. Carbamidomethylation of cysteine DAPI dissolved in H20 (0.01 g/mL, slices of 1–3 m thickness from formalin- was specified as fixed and oxidation of Sigma-Aldrich). Afterward, the coverslips fixed and paraffin-embedded tissue methionine as variable modification. One were dried and mounted with Aqua- blocks were deparaffinized. Endogenous missed trypsin cleavage was allowed. Poly/Mount (Polysciences, Warrington, peroxidases were blocked with 3% H2O2 The FDR for protein identification was PA, USA). All stainings were scanned by in PBS for 20 min followed by epitope set to 1% threshold. confocal microscopy (TCS SP5-II, Leica, blocking with 0.02% casein in PBS for 15 Flow cytometry. UT-7 cells were fixed Wetzlar, Germany). Illustration was done min. Immunoreaction was performed by with 4% Histofix solution (Carl Roth, using Imaris 7.5.1 (www.bitplane.com incubation with ctEPOR-AB (1:500, Karlsruhe, Germany). For EPOR staining, [Bitplane AG, Zurich, Switzerland]). Synaptic Systems) overnight at room cells were blocked, permeabilized with Immunohistochemistry on frozen temperature, followed by the addition of 5% normal horse serum (NHS) and 0.5% mouse sections. C57BL/6N mice, 5 wks the secondary biotinylated donkey anti- Triton X-100, and incubated with old, were anesthetized by i.p. injection rabbit AB (1:500; Amersham Biosciences, ctEPOR-AB (1:500, Synaptic Systems) and (0.276 mg/g tribromoethanol, Sigma- Freiburg, Germany) and an extravidin- Hoechst (5 μg/mL Invitrogen) or for con- Aldrich) and perfused transcardially with peroxidase enzyme complex (1:1000; trol only Hoechst for 30 min on ice. After 0.9% saline followed by 4% formaldehyde Sigma-Aldrich), each for 1 h at room tem- washing, cell suspensions were incubated in PBS. Brains were removed, postfixed perature. The AB reaction was visualized with Alexa Fluor 488 donkey anti-rabbit overnight at 4°C with 4% formaldehyde with the chromogen AEC: 4 mL 4% 3- (1:250, Life Technologies GmbH) for 30 in PBS and placed in 30% sucrose in PBS amino-9-ethylcarbazole (Sigma-Aldrich) min on ice, followed by FACS analysis for cryoprotection and stored at –20°C. in N,N-dimethylformamide (Merck) were (FACSAria III, BD Biosciences, Heidel- Whole mouse brains were cut into 30 μm- dissolved in 56 mL 0.1 mol/L sodium- berg, Germany). thick coronal sections on a cryostat acetate buffer adjusted to pH 5.2 with
Immunocytochemistry. Cells were (Leica). Frozen sections were permeabi- acetic acid and 1% H2O2. The brain slices fixed with 4% formaldehyde in PBS for 20 lized and blocked with 0.5% Triton X-100 were counterstained with hemalum. Cov- min, permeabilized and blocked in 0.2% and 5% NHS in PBS for 1 h at room tem- erslips were mounted with Aquatex Triton X-100 with 10% NHS in PBS for 20 perature. Then, sections were incubated (Merck). min. After washing with 1% NHS in PBS, with the following primary AB in 3% cells were incubated overnight at 4°C NHS, 0.5% Triton X-100 in PBS for 48 h at RESULTS with the primary AB in 0.2% Triton X-100 4°C: Rabbit ctEPOR-AB (1:200), chicken with 1% NHS in PBS. The following pri- anti-NeuN (266 006; 1:500), guinea pig Generation of EPOR-AB mary AB were used: rabbit ctEPOR-AB anti-GFAP (173 004; 1:500, all Synaptic The aim of this work was to generate (1:1000, Synaptic Systems), mouse Systems) and mouse anti-APC (clone CC- specific and sensitive EPOR-AB and to ntEPOR-AB (1:1000, Synaptic Systems), 1, 1:100, Merck). After washing in PBS, investigate EPOR expression in the cen- mouse anti-HA (1:500, Covance Inc., sections were incubated with the follow- tral nervous system (CNS). Therefore, Princeton, NJ, USA), mouse anti-GM130 ing secondary AB in 3% NHS, 0.5% Triton polyclonal rabbit EPOR-AB and mono-
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clonal mouse EPOR-AB were produced EOC-20 cells (N-terminally HA-tagged we performed IPs from UT-7 lysates and and tested. After extensive characteriza- human EPOR) and respective controls subsequent mass spectrometric protein tion of a whole panel of AB (data not showed specific detection of full-length identification. We used covalently immo- shown), two highly promising candi- human EPOR by ctEPOR-AB (Figure 1H). bilized ctEPOR-AB in combination with dates were selected and validated for This was validated with an HA im- nonreducing elution conditions to mini- several research purposes: The poly- munoblot of the same lysates (Figure 1I). mize the masking effect of excess anti- clonal rabbit EPOR-AB SA7378, directed In protein lysates from UT-7 and body heavy chains, which have an appar- against the C-terminus (here always OCIM-1 cells, ctEPOR-AB detected ent electrophoretic mobility similar to the called “ctEPOR-AB”) and the mono- bands of the same molecular weight (Fig- EPOR. After IP with ctEPOR-AB, eluted clonal mouse EPOR-AB 45A3, directed ure 1J). As shown in Figures 1F and J, in proteins from ctEPOR-AB protein-G against the N-terminus (here called UT-7 cells and HEK293 FT cells (only sepharose beads and respective control “ntEPOR-AB”). The present study is when transfected with the full length beads were separated by SDS-PAGE and mainly built on ctEPOR-AB because of human EPOR) a specific degradation visualized by colloidal Coomassie stain- its high specificity and broad spectrum product was additionally detected at ing or immunoblotting. The overlay of of applications in human and mouse. ~40kDa by the ctEPOR-AB. This degra- the two gel images (Figure 2A) was used dation product of EPOR has been de- to identify the region of the Coomassie- Functional EPOR Validation in the Test scribed earlier (11). Fixed UT-7 cells stained gel potentially containing the Systems were successfully used for flow cyto- EPOR protein. Identical gel regions from As a prerequisite of testing EPOR-AB metric analysis after staining with ctEPOR-AB IP and the control IP were ex- in the cell lines used here, we function- ctEPOR-AB (Figure 1K). EPOR was fur- cised and subjected to tryptic digestion ally validated their EPOR expression. In ther recognized by ctEPOR-AB in followed by liquid chromatography cou- the EPO-dependent megakaryoblastic human placenta and fetal brain extracts pled to mass spectrometry (LC-MS). leukemia cell line UT-7, incubation with (Figure 1L). In addition to human Against a common background mainly different concentrations of rhEPO led to EPOR, ctEPOR-AB detected murine consisting of chaperone proteins, human STAT-5 phosphorylation (Figure 1A). EpoR in transfected HEK293 FT cells, EPOR protein was detected in eluates Also, in the erythroleukemia cell line mouse fetal liver and lysates from cul- from ctEPOR-AB beads, but not from OCIM-1, incubation with rhEPO induced tured primary mouse oligodendrocytes control beads. The identification of 11 STAT-5 phosphorylation (Figure 1B). (Figure 1M). To validate the specificity EPOR-derived peptides with high mass UT-7 cells only proliferated and survived of murine EpoR detection, EpoR was accuracy at both precursor and fragment in the presence of rhEPO in the medium knocked out in primary astrocytes de- ion level resulted in sequence coverage of (Figures 1C, D). In the mouse microglia rived from EpoR-fl/fl mice. In fact, 22.6% (Figures 2B, C), basically in line cell line EOC-20, stably transduced with ctEPOR-AB recognized a doublet of with recent LC-MS data on EPOR im- N-terminally HA-tagged human EPOR, bands (EPOR with or without munoprecipitates (37). Taken together, rhEPO administration activated MAPK N-glycosylation, resulting in a differ- our results show that ctEPOR-AB indeed phosphorylation in a concentration-de- ence of ~3kDa) with a molecular weight binds specifically to full-length EPOR. pendent manner (Figure 1E). We also of around 65 kDa (Figure 1N, control Also, in reducing conditions we could ef- confirmed EPOR mRNA expression in all transduction). Shown is a clear reduction fectively elute EPOR after IP (Figure 2D). of these cell lines by qPCR (normalized upon expression of Cre-recombinase. The Noteworthy, we detected the EPOR pro- to GAPDH as housekeeping gene, data residual expression of the protein is tein in Western blots between 59 and 68 not shown). likely due to a slow turnover of EpoR, kDa, depending on the gel system and slow kinetics of Cre-recombination of the protein molecular weight marker used Detection of EPOR/EpoR by Western floxed EpoR allele or incomplete infec- (Figures 1, 2A, D; also see Materials and Blotting tion of the lentivirus. In any case, Cre- Methods). To confirm reliable detection of EPOR dependent reduction of the signal led us by Western blotting, we transfected to conclude that ctEPOR-AB specifically EPOR/EpoR Detection by HEK293 FT cells with different EPOR detects EpoR. Together, these results in- Immunocytochemistry expression vectors. The polyclonal rab- dicate specific EPOR/EpoR detection To validate the specificity of ctEPOR- bit ctEPOR-AB detected full-length with ctEPOR-AB in human and murine AB on formaldehyde fixed cells, we human EPOR and its degradation prod- cell and tissue extracts. stained the same antigen with AB di- uct specifically while the C-terminally rected against different epitopes (38). truncated mutant of EPOR was not de- EPOR Protein Identification In EOC-20 cells transduced with an tected by this AB (Figures 1F, G). Im- To test whether the specific band de- N-terminally HA-tagged human EPOR, munoblots of lysates from transduced tected in Western blots is indeed EPOR, anti-HA and ctEPOR-AB double-staining
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Figure 1. Functional EPOR validation and EPOR/EpoR detection using ctEPOR-AB in Western blots. (A) Incubation of EPO-dependent UT-7 cells (after 12 h of EPO deprivation) for 15 min with increasing EPO concentrations inducing STAT5 phosphorylation. (B) Incubation of OCIM-1 cells for 15 min with increasing EPO concentrations inducing STAT5 phosphorylation. (C) Cell counts of EPO-dependent UT-7 cul- tures 72 h after seeding in the presence and absence of EPO (n = 6, mean ± SEM; p < 0.0001). (D) Cell death in EPO-dependent UT-7 cul- tures 72 h after seeding in presence and absence of EPO (n = 5, mean ± SEM; p < 0.03). (E) Incubation of EOC-20 cells transduced with HA-tagged human EPOR for 10 min with increasing EPO concentrations inducing MAPK phosphorylation. (F) EPOR Western blot using ctEPOR-AB on transfected HEK293 FT cell lysates (truncated HA-EPOR: human EPOR lacking the C-terminus, HA-tag at the N-terminus; control vector: anchored human EPOR without N-terminus and C-terminus). (G) HA Western blot of the same transfected HEK293 FT cell lysates used in (F). (H) EPOR Western blot using ctEPOR-AB on EOC-20 cell lysates transduced with N-terminally HA-tagged human EPOR and respective controls. (I) HA Western blot of the same EOC-20 cell lysates used in (H). (J) EPOR Western blot using ctEPOR-AB on UT-7 and OCIM-1 cell lysates. (K) Flow cytometry of fixed UT-7 cells stained with ctEPOR-AB and Alexa Fluor 488 donkey anti-rabbit secondary AB; as control secondary AB only. (L) EPOR detection in human placenta and human fetal brain using ctEPOR-AB. (M) Detection of murine EpoR in transfected HEK293 FT cells overexpressing murine EpoR, mouse fetal liver and mouse primary oligodendrocytes using ctEPOR-AB. (N) Lentivirus-mediated conditional EpoR knockout in primary EpoR-fl/fl mouse astrocytes; anti-α tubulin as stably expressed comparator.
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Figure 2. EPOR IP using ctEPOR-AB and protein identification by mass spectrometry. Continued on next page
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Figure 2. Continued. (A) Colloidal Coomassie staining and immunoblot of the same EPOR IP using ctEPOR-AB from UT-7 protein lysates. The overlay was used to determine the region to be excised from the Coomassie gel for subsequent mass spectrometric protein identification (area indicated by rectangles; abbreviations: FT = flow-through, IP = immunoprecipitation). (B) Amino acid sequence of EPOR (UniProtKB/Swiss-Prot P19235). Peptides identified by mass spectrometry are indicated in red. Note that large parts of the EPOR precursor se- quence (indicated in italics) cannot be covered in a standard proteomic experiment with tryptic cleavage as they are either modified (amino acids 1–34, signal peptide; 57–89, N-glycosylation site), attached to the transmembrane domain (224–275) or too large (>5 kDa) to re- veal useful information by mass spectrometric sequencing (379–453, 454–508). (C) Table with details on peptide identification. Columns show from left to right: numbering of tryptic peptides; numbering of amino acids according to the sequence in B; peptide sequence (c, carbox- amidomethyl-Cys); observed and calculated mass of the singly protonated peptide; peptide mass deviation in ppm; PLGS score; number of b–y fragment ions; root mean square fragment mass deviation in ppm. (D) Immunoblot of EPOR IP using ctEPOR-AB from UT-7 lysates. In con- trast to the IP used for mass spectrometry, EPOR was eluted from the beads in reducing conditions (Laemmli buffer with β-mercaptoethanol; abbreviations: FT = flow-through, IP = immunoprecipitation, Ig HC = immunoglobulin heavy chains, Ig LC = immunoglobulin light chains). The prominent band at around 40 kDa in both IP conditions has to be an immunoglobulin fragment eluted from the beads in the reducing con- dition only, since it was not eluted without β-mercaptoethanol (see subpanel 2A immunoblot). showed almost complete colocalization injections in NG2-CreERT2 mice (28). At double labeling with HA-AB and with most of the signal located intracel- 72 h after the second tamoxifen injection, ctEPOR-AB confirmed specific recovery lularly (Figure 3A). Control EOC-20 cells we identified precursors double-stained of these cells in frozen sections of were negative (Figure 3A). In UT-7 cells, for GFP and ctEPOR-AB (GFP+/EpoR+), paraformaldehyde-perfused mice. Also in ctEPOR-AB revealed a similar staining as well as GFP+/EpoR+ cells with clear this condition, endogenous cells with pattern (Figure 3B). EPOR staining was morphology (processes with parallel high EpoR expression were observed in colocalized with Golgi staining, indicat- myelin bundles) of already differentiated close proximity of the injection site (Fig- ing detection of a membrane protein oligodendrocytes (Figures 4B–B’’). More- ures 4H–H”). These results confirm the (Figure 3C). In addition, ntEPOR-AB over, GFAP+/EpoR+ cells were seen in pronounced upregulation of EpoR in cells and ctEPOR-AB double-stained EPOR in postnatal neurogenesis areas such as den- reacting to injury, provoked here by an UT-7 cells (Figure 3D). In OCIM-1 cells, tate gyrus (Figure 4C) or subventricular experimental stab wound. ctEPOR-AB also yielded intracellular zone (data not shown). These results indi- staining, even though less pronounced cate EpoR expression in differentiating Upregulation of EPOR in the compared with UT-7 cells (Figure 3E). oligodendrocytes and stem cells in the Hippocampal Formation of a Patient Using ctEPOR-AB, we also detected adult neurogenic niches of healthy young Suffering from Temporomesial EPOR in human IPS cells (Figure 3F). mice. Complex-Focal Epilepsy Moreover, ctEPOR-AB specifically Formalin-fixed, paraffin-embedded tis- stained HEK293 FT cells transfected EPOR/EpoR Detection in the Injured sue from a patient who underwent selec- with full-length murine EpoR (Figure CNS of Mice tive unilateral hippocampectomy, was 3G). When tested on cultured primary Next, we stereotactically injected used for immunohistochemical detection murine brain cells, ctEPOR-AB stained EPOR-transduced EOC-20 cells or me- of EPOR upregulation under these condi- oligodendrocyte precursor cells (Figure dium only (stab wound analogue) in the tions (Figure 5A). The patient had been 3H), oligodendrocytes (Figure 3I) and motor cortex of adult mice (Figure 4D). suffering from pharmacoresistant microglia (Figure 3J). These results indi- This experiment served two purposes: to complex-focal seizures of temporomesial cate specific staining of human and recover defined cells that carry human origin for more than 10 years. Neu- mouse EPOR/EpoR by the ctEPOR-AB EPOR in brain sections and to confirm ropathological analysis of the surgery in cell lines and primary cells. injury-induced endogenous EpoR expres- material revealed hippocampal sclerosis sion, since in earlier work, we had pro- stage Wyler III. EPOR was upregulated EpoR Detection in the Brain of Healthy posed upregulation of EPOR upon injury in several but not all remaining neurons Mice (6). At 24 h after injection of medium of CA1 (Figure 5B), of CA4 (Figure 5C) Using ctEPOR-AB on frozen brain sec- only (stab wound), we saw cells with and of the dentate gyrus (Figure 5D), as tions of healthy young mice, we found strong ctEPOR-AB signal near the injec- well as in oligodendrocytes and endothe- EpoR expression mainly in a subpopula- tion site (Figure 4E). Many of these cells lial cells of capillaries in the adjacent tion of cells of the oligodendrocyte line- were GFAP+/EpoR+ (Figure 4F). On the white matter (Figure 5E). Without pri- age (Figure 4A). To get better insight at contralateral site, no GFAP+/EpoR+ cells mary AB, no staining could be detected which stages cells of the oligodendrocyte were seen (Figure 4G). In the motor cor- (Figure 5F). This suggests upregulation lineage express EpoR, we labeled oligo- tex of mice injected with transduced of EPOR upon severe chronic distress in dendrocyte precursor cells by tamoxifen EOC-20 cells (murine microglia cell line), different cell types of the human CNS.
MOL MED 21:803-815, 2015 | OTT ET AL. | 811 EPOR IN BRAIN
Figure 3. Detection of EPOR by immunocytochemistry: (A) EPOR detection using ctEPOR-AB and monoclonal HA-AB on transduced (N- terminally HA-tagged human EPOR, upper row) and control (lower row) EOC-20 cells. (B) EPOR staining with ctEPOR-AB on EPO-depen- dent UT-7 cells. (C) Double-immunostaining of UT-7 cells with anti-GM130 AB as marker for the Golgi apparatus and ctEPOR-AB. (D) EPOR double staining of UT-7 cells with ntEPOR AB and ctEPOR-AB. (E) EPOR staining with ctEPOR-AB on OCIM-1 cells. (F) Distinct EPOR staining in human Oct-4 + IPS cells using ctEPOR-AB. (G) EpoR staining with ctEPOR-AB of HEK293 FT cells transfected with full-length murine EpoR. Neighboring nontransfected cells show no immunofluorescence. (H) EpoR and NG2 double-staining with ctEPOR-AB in primary mouse oligodendrocyte precursor cells. (I) EpoR and CC-1 double-staining of primary mouse oligodendrocytes with ctEPOR-AB. (J) Detection of EpoR in primary mouse microglia using ctEPOR-AB and lectin as counterstain.
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Figure 4. EPOR detection using ctEPOR-AB in healthy and injured mouse brain by immunohistochemistry: (A) EpoR staining in a subpopula- tion of CC-1 positive mature oligodendrocytes in the neocortex of a 5-wk-old healthy mouse. (B, B’, B’’) EpoR staining in the hippocampus of a 5-wk-old NG2-CreERT2:R26-td-tomato-mEGFP mouse. Some oligodendrocyte precursor cells (arrow head) and newly differentiated oligodendrocytes (arrow) express EpoR. Both cell types are endogenously labeled with membrane-tagged EGFP. (C) EpoR staining of GFAP+ cellular processes in the dentate gyrus of a 5-wk-old mouse (arrow heads). (D) Overview of the injection site in the motor cortex of an 8-wk-old mouse injected with medium only (stab wound analogue). The section was stained for neuronal nuclei with NeuN and for EpoR with ctEPOR-AB. (E) Close-up of the white-rectangle region in (D) shows reactive cells with upregulated EpoR expression. (F) Many of the cells at the injection site with upregulated EpoR expression are GFAP+ (arrow heads). (G) Contralateral to the injection site, GFAP+ cells show no EpoR expression at 24 h after lesion. (H, H’, H’’) Shown is EPOR and HA double-labeling of injected EOC-20 microglial cells trans- duced with an HA-tagged human EPOR. In addition, HA-negative cells at the injection site show strong EpoR expression (arrow heads).
DISCUSSION polyclonal rabbit AB directed against the flow cytometry and IP to immunocyto- In the present work, we took the chal- intracellular C-terminus of the human chemistry and immunohistochemistry on lenge requested for a long time by the EPOR. This AB, referred to as “ctEPOR- frozen as well as paraffin-embedded sec- scientific community (14,17–18) to gener- AB,” specifically recognizes EPOR, as tions. Importantly, by employing this AB, ate a specific AB for valid detection of proven by mass spectrometry, and has a we were able to confirm expression of EPOR in human and murine cells and broad range of documented applications EPOR in brain cells and its upregulation tissues, with particular focus on the in both human and murine cells and tis- upon injury (39). Our comprehensive in brain. We present here a highly specific sues, ranging from Western blotting, vitro and in vivo data clearly reject earlier
MOL MED 21:803-815, 2015 | OTT ET AL. | 813 EPOR IN BRAIN
CONCLUSION On the basis of the novel tool re- ported here, it will now be possible to investigate the role of EPOR in the in- tact and injured human and murine brain in more detail. This, in turn, will facilitate the development of EPO for therapeutic use outside the hematopoi- etic system.
ACKNOWLEDGMENTS This work was supported by ZIM- BWMi, the Max Planck Society, the Deutsche Forschungsgemeinschaft (Cen- ter for Nanoscale Microscopy and Molec- ular Physiology of the Brain, SFB- TRR43), EXTRABRAIN EU-FP7 and the Figure 5. EPOR upregulation in neurons and oligodendrocytes of the hippocampal forma- Niedersachsen-Research Network on tion of a patient suffering from temporomesial complex-focal epilepsy as demonstrated Neuroinfectiology (N-RENNT). The by immunohistochemistry using ctEPOR-AB. (A) Overview of the surgical sample stained EPO-dependent megakaryoblastic with ctEPOR-AB. (B) Upregulation of EPOR, visualized by brown color of the AEC-chro- leukemia UT-7 cell line was a kind gift mogen, in some (arrows) but not all (arrow heads) neurons of the CA1 region. (C) EPOR from Drorit Neumann of Tel Aviv Uni- positive neurons in the CA4 region (arrows). (D) Dentate gyrus neurons (arrows) expressing versity in Israel. The pMOWS vector, EPOR. (E) EPOR positive oligodendrocytes (arrows) as well as endothelial cells of capillar- encoding N-terminally HA-tagged full- ies (arrow heads) in the adjacent white matter. (F) No staining was observed when omit- length human EPOR plus puromycin- ting the primary antibody. resistant cassette, was a kind gift from Ursula Klingmüller, DKFZ, Heidelberg, claims, solely based on in vitro studies, for generating specific mouse mono- Germany. The rat anti-NG2 was a kind that EPOR expression and EPO function clonal AB. They will be tested alone or gift from Jacqueline Trotter, University of outside the hematopoietic system does in the form of potentially more sensitive Mainz, Germany. We thank Marina not exist (15). cocktails, with collective properties simi- Uecker and Thomas Liepold for their ex- The great need for specific EPOR-AB lar to ctEPOR-AB. pert technical help. in the field is also reflected by a very re- With the examples of EPOR expression cent study of Drorit Neumann and col- in the brain shown here, we confirmed DISCLOSURE leagues (37). This group of authors pub- earlier work of our own and of others, H Stadler is member of the board of lished specific mouse and rat which in the past needed additional and holds stocks in Synaptic Systems monoclonal EPOR-AB that detect EPOR methods for validation and still left GmbH. H Martens, C Erck and T Kolbow expression in human cancer cells and tis- doubts in the scientific community due to are full-time employees of Synaptic Sys- sues (37). Complementary to this ap- the nonspecificity of previous EPOR-AB. tems GmbH. proach and with particular focus on the For instance, GFAP-positive stem cells in brain, we have developed a highly spe- the adult neurogenic niches showed REFERENCES cific and sensitive polyclonal rabbit AB, EpoR immunoreactivity here, which is in 1. Brines M, Cerami A. (2005) Emerging biological ctEPOR-AB, suitable for applications not line with reports demonstrating distinct roles for erythropoietin in the nervous system. only in human but also in murine mate- effects of EPO on adult neural stem cells Nat. Rev. Neurosci. 6:484–94. 2. Jelkmann W. (2007) Erythropoietin after a century rial. Since the polyclonal nature of this (40–41). Also, studies identifying EPO as of research: younger than ever. Eur. J. Haematol. AB has limitations, not only due to the an inducer of oligodendrocyte precursor 78:183–205. restricted lifetime of a rabbit, we are cur- cell differentiation (42–43) are now fur- 3. Jelkmann W. (2013) Physiology and pharmacol- rently working on further exploitation of ther supported by the detection of spe- ogy of erythropoietin. Transfus. Med. Hemother. the herewith acquired knowledge. Pre- cific EPOR-binding sites in culture and 40:302–9. 4. Arcasoy MO. (2008) The non-haematopoietic bio- liminary results of epitope mapping brain sections. Importantly, the role of the logical effects of erythropoietin. Br. J. Haematol. with this polyclonal ctEPOR-AB re- EPO/EPOR system in response to brain 141:14–31. vealed only few strongly recognized epi- injury (5–6) is confirmed with our stab 5. Sargin D, Friedrichs H, El-Kordi A, Ehrenreich H. topes. These epitopes are presently used wound approach. (2010) Erythropoietin as neuroprotective and neu-
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APPENDIX
Manuscript under revision
Manuscript 1
Oliveira B*, Mitjans M*, Nitsche MA, Kuo M-F and Ehrenreich H. Excitation-inhibition dysbalance as predictor of autistic phenotypes. Under revision
*Authors with equal contribution
Personal contribution: I actively participated in outlining and writing the manuscript, together with my supervisor. I was significantly involved in the data analysis, prepared the figures and figure legends and contributed to the overall preparation of the manuscript. Additionally, I was responsible for the submission phase and will actively participate in the revision process.
145
Excitation-inhibition dysbalance as predictor of autistic phenotypes
Bárbara Oliveira, MSc1§, Marina Mitjans, PhD1§, Michael A. Nitsche, MD2, Min-Fang Kuo, MD, PhD2* and Hannelore Ehrenreich, MD, DVM1*
§Equal contributing first authors *Equal contributing last authors
1Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany 2Department of Psychology and Neurosciences, Leibniz Research Centre for Working Environment and Human Factors, Dortmund, Germany
Word count: Abstract 250; Text body 1204; 1 Figure; 1 Table
Running head: E/I dysbalance and autistic traits
Key words: TMS, transcranial magnetic stimulation, behavioral continua, severity rating, PAUSS, autistic traits
Corresponding Author: Prof. Hannelore Ehrenreich, MD, DVM Clinical Neuroscience Max Planck Institute of Experimental Medicine Hermann-Rein-Str.3 37075 Göttingen, GERMANY Tel: 49-551-3899 628 E-Mail: [email protected]
1
Abstract
Autistic traits are normally distributed across health and disease, with autism spectrum disorders (ASD) at the extreme end. As we learned from mutations of synaptic or synapse regulating genes, leading to monogenetic forms of autism, the heterogeneous etiologies of ASD converge at the synapse. They result in a mild synaptic dysfunction as the final common pathway, also addressed as synaptopathy. Based on genetic rodent models and EEG/MEG findings in autists, a neuronal excitation-inhibition dysbalance is considered autism-pathognomonic. We hypothesized that this objectively measurable consequence is not restricted to the diagnosis of ASD but transcends disease borders and is of quantitative rather than qualitative nature. For proof-of-principle, we conducted a transcranial magnetic stimulation (TMS) study, monitoring corticospinal excitability and intracortical inhibition of the motor cortex. Employing the GRAS data collection of N>1800 deep-phenotyped schizophrenic subjects, we had the chance to select for this study N=20 perfectly matched men. They differed only by autistic trait severity, as assessed using PANSS autism severity score (PAUSS), capturing the continuum of autistic behaviors. Applying TMS to these men, we provide intriguing hints of a positive correlation of autistic phenotype severity with functional cortical correlates, mainly alterations in GABAergic system and ion channels. This ‘dose-response relationship’ between severity of autistic traits and excitation-inhibition ratio in non-ASD subjects underlines the biological basis of this continuous trait. Based on these data, TMS may serve as new add-on biomarker of autistic traits across disease groups. Moreover, potential common treatment strategies targeting the excitation-inhibition dysbalance in humans may now evolve.
2
Introduction Most neuropsychiatric disease-relevant behavioral phenotypes are continua, with a certain severity threshold defining the starting point of a clinical diagnosis. As typical example, autistic traits are normally distributed across health and disease, including at the extreme end autism spectrum disorders (ASD). Shared features are variably pronounced deficits in social communication, reading of social signals, theory-of-mind abilities, and cognitive flexibility, typically together with restricted interests, repetitive behaviors and prominent routines (Mitjans et al., 2017; Stepniak et al., 2015).
Causes of autistic phenotypes likely converge at the synapse, as indicated by mutations of genes influencing synaptic function, and are reflected by circuit-level disturbances, aberrant synaptic plasticity, and a virtually autism-pathognomonic neuronal excitation- inhibition dysbalance (Mitjans et al., 2017; Mullins et al., 2016; Nelson and Valakh, 2015; Ramocki and Zoghbi, 2008; Sudhof, 2008; Uhlhaas and Singer, 2006; Uzunova et al., 2016). This dysbalance was consolidated experimentally using construct-valid genetic rodent models and has likewise been suspected in humans, mainly based on EEG and MEG findings, including the frequently seen predisposition to epileptic seizures. Importantly, not only mutations, but normal genetic variants contribute to the manifestation of autistic traits as indicated by genome-wide association studies (GWAS), but even more so by phenotype-based genetic association studies (PGAS), finding an accumulation of ‘unfortunate’ normal variants associated with increasing severity of autistic phenotypes (Ehrenreich et al., 2016; Stepniak et al., 2015).
We hypothesize that measurable consequences of the heterogeneous causes underlying autistic traits, which characterize the final common pathway to the phenotype, are of quantitative rather than qualitative nature. Thus, we wondered whether very well-matched individuals, differing only with regard to the clinical severity of autistic features, would show respective differences in excitation-inhibition as an overarching functional consequence of diverse causalities. To address this question in humans, we applied transcranial magnetic stimulation (TMS) (Nitsche et al., 2005). TMS, presently discussed as ASD biomarker tool, allows noninvasive focal brain stimulation, where localized intracranial electrical currents, large enough to depolarize a small population of neurons, are generated by extracranial magnetic fields (Oberman et al., 2016). Translating this paradigm to the autistic continuum, we provide intriguing
3 evidence of a connection between phenotype severity and functional cortical correlates in humans.
Materials and methods Subjects
For proof-of-principle, carefully selected male schizophrenic subjects (N=20) of the GRAS (Göttingen Research Association for Schizophrenia) data collection agreed to participate in a TMS study, approved by the Ethical Committee of the University of Göttingen, Germany. Subjects were matched for age, handedness and medication and asked not to smoke or drink coffee prior to TMS (Table1). For comparing deeply phenotyped schizophrenic individuals, based on autistic trait severity, we used the PANSS autism severity score (PAUSS), capturing the continuum of autistic behaviors.12
Transcranial magnetic stimulation (TMS) protocol Motor cortex excitability measures
Motor cortical excitability was monitored by generating motor evoked potentials (MEP) via TMS applied with a figure-of-eight coil (diameter of one winding: 70mm; peak magnetic field: 2.2Tesla) connected to a Magstim-200 magnetic stimulator (Magstim, Whiteland, Dyfed, UK). The coil was held with the handle pointing backwards and laterally at 45°. Surface electromyography (EMG) was recorded from the right first dorsal interosseous (FDI) muscle by Ag/AgCl electrodes in a belly tendon montage. The signals were filtered with a low-pass filter of 2.0kHz, then digitized at an analogue-to- digital rate of 5kHz and further relayed into a laboratory computer using the Signal software and CED1401 hardware (Cambridge Electronic Design, Cambridge, UK). Motor threshold
The resting motor threshold (RMT) was defined as the minimum TMS intensity which elicited a peak-to-peak MEP amplitude of 50µV or larger in the resting FDI in at least 3 out of 6 measurements. The active motor threshold (AMT) was the minimum intensity eliciting a MEP of a superior size compared to moderate spontaneous muscular background activity (∼15% of the maximum muscle strength) in at least 3 out of 6 trials.
4
Input-output (I/O) curve
The recruitment curve was generated with TMS-intensities of 100, 110, 130, and 150% RMT; 15 stimuli were obtained for each intensity. TMS stimuli were applied with a frequency of 0.25Hz. Intracortical inhibition and facilitation
Intracortical inhibition and facilitation were obtained by a paired-pulse TMS protocol (Kujirai et al., 1993), with a test pulse eliciting MEP amplitude of about 1mV. MEP was preceded by a subthreshold (70% AMT) conditioning stimulus with inter-stimuli-intervals (ISI) of 2, 3, 5, 10, and 15ms. The first three ISIs monitor intracortical inhibition and the last two monitor facilitation. The protocol consisted of 15 blocks, where each block contained the 5 pairs of different ISIs together with a single test pulse applied in randomized order. TMS stimuli were applied with an interval of 4 seconds. Experimental procedures
Subjects were seated in a reclining chair with head and arm rests to guarantee optimal relaxation. After mounting the EMG electrodes, the TMS coil position over the left motor cortex resulting consistently in the largest MEP in the right FDI was identified and marked to keep constant coil position throughout the course of the experiment. The intensity of the TMS stimulus was adjusted to elicit MEPs with a peak-to-peak amplitude of average 1 mV. RMT and AMT were then determined accordingly, followed by a 5-min break to allow muscle relaxation after the AMT determination procedure. Afterwards, the I/O curve, and intracortical inhibition/facilitation were obtained as outlined above. The duration of this experimental block was about 60 minutes per individual.
Results RMT and AMT did not differ between groups (low PAUSS: 42.50±5.33 versus high PAUSS: 44.00±9.16; p=0.909 and low PAUSS: 32.90±4.43 versus high PAUSS: 34.90±7.38; p=0.622, respectively; Mann-Whitney test, two-tailed). Individual excitation and inhibition curves are presented in Fig 1A-B. The group of schizophrenic men with more severe autistic features (PAUSS≥20) showed higher cortico-spinal excitability and higher intracortical inhibition compared to the group with low autistic features (PAUSS<20). In contrast, intracortical facilitation did not differ between groups (Fig 1A-
5
B; Table1). Importantly, the ratio between excitation and inhibition (each calculated as area under the curve for each individual) correlated positively with the severity of autistic traits (r=0.511, p=0.021; Pearson’s Correlation, two-tailed) (Fig 1C).
Discussion Even though only 20 male schizophrenic subjects with different PAUSS scores were included, we obtained a surprisingly clear separation of TMS-derived individual excitation and inhibition readouts based on PAUSS and, importantly, a positive correlation of the E/I ratio with PAUSS. These findings provide for the first time a ‘dose- response relationship’ between severity of autistic traits and degree of excitation- inhibition dysbalance. The clarity of these results is certainly owed to an optimal matching of deep-phenotyped individuals regarding all main clinical characteristics, based on selection from the large GRAS data collection, which allows widely excluding potential confounders (Oberman et al., 2016).
We interpret these data as indication of effects on GABAergic transmission and ion channels, and no clear effect on glutamate, since intracortical inhibition is primarily driven by GABA (Paulus et al., 2008). The enhanced corticospinal excitability shown in the group with high autistic features could principally have been driven by an impact on ion channels, but also by glutamate, especially for the high TMS intensities (Paulus et al., 2008). Since glutamate-driven intracortical facilitation did, however, not differ between groups, a predominant impact on ion channels is more probable. Taken together, the results of this study support the concept of a disturbed excitation-inhibition balance in subjects with autistic features, which is based on altered GABAergic and ion channel functions.
These findings support the disease-independent pathophysiological continuum of autistic traits and the etiology-independent final common pathway leading to an autistic phenotype. Not only the diagnostic use of TMS as a new add-on biomarker of autistic traits across disease groups, but also potential common treatment strategies targeting the E/I ratio in humans should now be encouraged.
6
Acknowledgements This work was supported by the Max Planck Society, the Max Planck Förderstiftung, the DFG (CNMPB), EXTRABRAIN EU-FP7, the Niedersachsen-Research Network on Neuroinfectiology (N-RENNT) and EU-AIMS. The research of EU-AIMS receives support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n°115300, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013), from the EFPIA companies, and from Autism Speaks. The authors thank all subjects for participating in the study, and the many colleagues who have contributed over the past decade to the extended GRAS data collection.
AUTHOR CONTRIBUTIONS Concept and design of the study: HE, MFK Data acquisition and analysis: BO, MM, MFK, MAN, HE Drafting manuscript and figures: HE, BO, MM, with help of all authors All authors read and approved the final version of the manuscript.
Competing interests The authors declare no conflict of interest.
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Ehrenreich, H., Mitjans, M., Van der Auwera, S., Centeno, T.P., Begemann, M., Grabe, H.J., Bonn, S., Nave, K.A., 2016. OTTO: a new strategy to extract mental disease-relevant combinations of GWAS hits from individuals. Mol Psychiatry. Kujirai, T., Caramia, M.D., Rothwell, J.C., Day, B.L., Thompson, P.D., Ferbert, A., Wroe, S., Asselman, P., Marsden, C.D., 1993. Corticocortical inhibition in human motor cortex. J Physiol 471, 501-519. Mitjans, M., Begemann, M., Ju, A., Dere, E., Wustefeld, L., Hofer, S., Hassouna, I., Balkenhol, J., Oliveira, B., van der Auwera, S., Tammer, R., Hammerschmidt, K., Volzke, H., Homuth, G., Cecconi, F., Chowdhury, K., Grabe, H., Frahm, J., Boretius, S., Dandekar, T., Ehrenreich, H., 2017. Sexual dimorphism of AMBRA1-related autistic features in human and mouse. Transl Psychiatry 7(10), e1247. Mullins, C., Fishell, G., Tsien, R.W., 2016. Unifying Views of Autism Spectrum Disorders: A Consideration of Autoregulatory Feedback Loops. Neuron 89(6), 1131-1156. Nelson, S.B., Valakh, V., 2015. Excitatory/Inhibitory Balance and Circuit Homeostasis in Autism Spectrum Disorders. Neuron 87(4), 684-698. Nitsche, M.A., Seeber, A., Frommann, K., Klein, C.C., Rochford, C., Nitsche, M.S., Fricke, K., Liebetanz, D., Lang, N., Antal, A., Paulus, W., Tergau, F., 2005. Modulating parameters of excitability during and after transcranial direct current stimulation of the human motor cortex. J Physiol 568(Pt 1), 291-303. Oberman, L.M., Enticott, P.G., Casanova, M.F., Rotenberg, A., Pascual-Leone, A., McCracken, J.T., Group, T.M.S.i.A.C., 2016. Transcranial magnetic stimulation in autism spectrum disorder: Challenges, promise, and roadmap for future research. Autism Res 9(2), 184-203. Paulus, W., Classen, J., Cohen, L.G., Large, C.H., Di Lazzaro, V., Nitsche, M., Pascual-Leone, A., Rosenow, F., Rothwell, J.C., Ziemann, U., 2008. State of the art: Pharmacologic effects on cortical excitability measures tested by transcranial magnetic stimulation. Brain Stimul 1(3), 151-163. Ramocki, M.B., Zoghbi, H.Y., 2008. Failure of neuronal homeostasis results in common neuropsychiatric phenotypes. Nature 455(7215), 912-918. Stepniak, B., Kastner, A., Poggi, G., Mitjans, M., Begemann, M., Hartmann, A., Van der Auwera, S., Sananbenesi, F., Krueger-Burg, D., Matuszko, G., Brosi, C., Homuth, G., Volzke, H., Benseler, F., Bagni, C., Fischer, U., Dityatev, A., Grabe, H.J., Rujescu, D., Fischer, A., Ehrenreich, H., 2015. Accumulated common variants in the broader fragile X gene family modulate autistic phenotypes. EMBO Mol Med 7(12), 1565-1579. Sudhof, T.C., 2008. Neuroligins and neurexins link synaptic function to cognitive disease. Nature 455(7215), 903- 911. Uhlhaas, P.J., Singer, W., 2006. Neural synchrony in brain disorders: relevance for cognitive dysfunctions and pathophysiology. Neuron 52(1), 155-168. Uzunova, G., Pallanti, S., Hollander, E., 2016. Excitatory/inhibitory imbalance in autism spectrum disorders: Implications for interventions and therapeutics. World J Biol Psychiatry 17(3), 174-186.
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Figure Legend Excitation and inhibition dysbalance dependent on autistic phenotype severity (A) Input-output curves as a readout of cortico-spinal excitability are presented for each individual, obtained with TMS-intensities of 100, 110, 130, and 150% RMT (15 stimuli for each intensity with a frequency of 0.25Hz). The severity of autistic traits determined by PAUSS is dichotomously classified for illustration in the figure. (B) Individual intracortical inhibition curves presented analogously to (A). Intracortical inhibition was obtained by a paired-pulse TMS protocol, MEP was preceded by a subthreshold (70% AMT) conditioning stimulus with ISI of 2, 3 and 5ms. (C) Individual E or I values were determined as areas under the curve (of individual data shown in A and B) and used to calculate the individual excitation/inhibition (E/I) ratios. Dysbalance is visualized in individuals with higher PAUSS scores. TMS, Transcranial Magnetic Stimulation; RMT, Resting Motor Threshold; MEP, Motor-Evoked Potential; AMT, Active Motor Threshold; ISI, Inter-Stimuli-Intervals; PAUSS, PANSS autism severity score (Mitjans et al., 2017; Stepniak et al., 2015); AU, Arbitrary Units; Pearson’s correlation, two-tailed p-value (SPSS version-17.0 IBM-Deutschland GmbH, Munich, Germany).
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Table 1: SUBJECT CHARACTERIZATION
Subject Age at Type of Smoker Age of disease Duration of Premorbid Excitation Inhibition Facilitation PAUSS CPZE ID* TMS (y) medication (cig/day) onset (y) disease (y) IQ (MWTB) (AUC, AU) (AUC, AU) (AUC, AU) Low PAUSS (<20) COFO 8 37 Atypical 600 Yes (40) 20 16 143 82.91 3.11 5.42 SEKO 8 31 Atypical 200 No 22 10 95 26.36 3.26 5.86 JOKR 8 38 Atypical 4 Yes (20) 32 6 85 12.99 3.07 6.62 JORI 9 29 Atypical 450 Yes (20) 17 12 107 57.68 3.61 9.48 CLLA 10 29 Atypical 375 No 23 6 130 31.08 2.96 5.96 KARI 10 31 Atypical 133 Yes (20) 22 9 112 169.86 2.44 3.65 BEZI 11 32 Atypical 1200 Yes (20) 19 13 92 63.48 2.97 5.55 MAMA 11 43 Atypical & typical 163 Yes (20) 31 12 92 25.01 2.18 6.12 SEBO 15 32 Atypical & typical 1956 Yes (15) 18 14 97 38.92 4.02 7.16 MIBO 17 31 No neuroleptics - Yes (20) 14 16 97 99.07 3.33 4.57 Mean 10.70 33.20 564.53 21.69 11.51 105.00 60.73 3.09 6.03 - - ±SD ±3.06 ±4.45 ±630.97 ±5.58 ±3.64 ±18.52 ±47.12 ±0.53 ±1.56
High PAUSS (≥20) MASC 20 28 Atypical 150 No 19 8 78 51.36 2.59 5.28 RERE 23 55 Atypical 67 Yes (2) 49 6 124 94.11 3.52 5.16 JURU 24 30 No neuroleptics - No 17 12 97 169.55 2.59 5.52 HAKA 27 30 Atypical & typical 1631 Yes (20) 18 12 91 59.10 2.25 4.83 GEFA 28 33 Atypical & typical 2491 Yes (20) 21 13 100 64.26 3.71 4.67 TYBA 29 31 Atypical 1067 Yes (20) 18 13 94 84.87 1.17 3.79 THFI 29 45 Atypical & typical 1110 Yes (40) 36 9 100 138.75 1.19 6.64 BJWA 30 35 Atypical 267 Yes (26) 23 12 92 184.80 2.22 5.70 MASI 31 32 Typical 168 No 25 7 93 62.50 3.34 7.73 MAGU 39 53 Atypical 300 No 19 34 130 122.49 2.52 4.31 Mean 28.00 37.26 805.55 24.62 12.64 99.90 103.18 2.51 5.36 - - ±SD ±5.19 ±10.23 ±837.51 ±10.33 ±7.97 ±15.63 ±48.21 ±0.87 ±1.14 Low versus high PAUSS 0.00001° 0.529° 0.620# 0.730° 0.628# 0.684° 0.684° 0.579° 0.031§ 0.044§ 0.143§ p-values
Notes: *Subject ID scrambled; °Mann-Whitney U-Test (two-tailed); #Fisher’s exact test (two-tailed); §Student t-test (one-tailed); PAUSS, PANSS autism severity score;1 2 TMS, Transcranial Magnetic Stimulation; MWTB, Mehrfachwahl-Wortschatz-Intelligenztest-B; CPZE, Chlorpromazine Equivalent; AUC, Area Under the Curve; AU, Arbitrary Units; cig, cigarettes; y, year
APPENDIX
List of abbreviations
Abbreviation Extended name
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid APC Antigen Presenting Cells ApoE Apolipoprotein E ASD Autism Spectrum Disorders BBB Blood Brain Barrier BCR B Cell Receptor BMP Bone Morphogenic Protein Caspr2 Anti-Contactin-Associated Protein-like 2 CCL-2 CC-chemokine Ligand c-MYC MYC Proto-oncogene CNS Central Nervous System CSF Cerebrospinal Fluid CTD Carboxy-Terminal Domain CXCR4 C-X-C Chemokine Receptor Type 4 DAPT N-[N-3,5-Difluorophenacetyl-L-alanyl]-S-phenylglycine t-butyl ester DCX+ Doublecortin DNA Deoxyribonucleic Acid EMX1 Empty Spiracles Homeobox 1 E/I Excitation/Inhibition Fc Fragment Crystallisable FGF2 Fibroblast Growth Factor 2 FGF8 Fibroblast Growth Factor 8 FITC Fluorescein Isothiocyanate FoxA2 Forkhead Box Protein A2 GABA γ-Aminobutyric Acid
GABAAR GABA Type A Receptor
GABABR GABA Type B Receptor GRAS Göttingen Research Association for Schizophrenia HLA Human Leukocyte Antigen Ig Immunoglobulins IgG Immunoglobulin G IL-1β Interleukin-1β
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IL-17A Interleukin-17A IPS Induced Pluripotent Stem Cell KA Kainate KLF4 Kruppel-Like Factor 4 LBD Ligand-Binding Domain LIN28 Zinc Finger CCHC Domain-containing Protein 1 LIM1 LIM homeobox 1 MEP Motor Evoked Potentials mGluR Metabotropic Glutamate Receptors MHC Histocompatibility Complex MK-801 Dizocilpine NFIA Nuclear Factor I A NMDA N-methyl-D-aspartate NMDAR NMDA Receptors NMDAR-AB NMDA Receptors Autoantibodies NTD Amino-Terminal Domain OTX1 Orthodenticle Homeobox 1 OCT4 Octamer-binding Transcription Factor 4 PANSS Positive and Negative Syndrome Scale PAUSS PANSS Autism Severity Score PAX6 Paired Box 6 qPCR Quantitative Real Time Polymerase Chain Reaction RNA Ribonucleic Acid SDF-1 Stromal Cell-derived Factor 1 SeV Sendai Virus SSEA4 Stage-Specific Embryonic Antigen-4 SOX2 Sex Determining Region Y-box 2 TCR T Cell Receptors TGF-β Transforming Growth Factor β TLR4 Toll-like Receptor 4 TMD Transmembrane Domain TMS Transcranial Magnetic Stimulation TNF-α Tumour Necrosis Factor α
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Curriculum vitae
Bárbara Oliveira Phone number: +49 17661488577 email: [email protected] EDUCATION
PHD IN SYSTEMS NEUROSCIENCE Institution: Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences GGNB; PhD program ‘Systems Neuroscience’ Thesis topic: “Genetic and autoimmune modulators of brain function in neuropsychiatric illness and health” Since 2014
MSC IN MOLECULAR BIOLOGY AND CYTOMICS IN HEALTH SCIENCES Institution: Northern Polytechnic Institute of Health, Porto, Portugal Thesis topic: “X-linked Intellectual Disability: Retrospective study of risk populations resorting to a set of genes implicated in intellectual disability” Grade: Excellent 2008-2011
BSC IN ANATOMICAL PATHOLOGY Institution: Northern Polytechnic Institute of Health, Porto, Portugal Grade: 16 in a scale of 0-20 2004-2008
PROFESSIONAL ACTIVITIES
PhD candidate at Clinical Neuroscience Institution: Max Planck Institute for Experimental Medicine, Göttingen, Germany Since 2014
PhD candidate at Neurogenetics and Mental Health Unit Institution: National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal 2012-2014
Research assistant at Neurogenetics and Mental Health Unit Institution: National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal 2011
Master student at Molecular Genetics Unit Institution: Center for Medical Genetics Jacinto de Magalhães, Porto, Portugal 2010-2011
Teaching assistant at Anatomical Pathology Department Institution: Northern Polytechnic Institute of Health, Porto, Portugal 2010
Anatomical Pathology technician at IPATIMUP Institution: Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal 2009
AWARDS AND FELLOWSHIPS
• Early Stage Researcher fellowship, Marie Skłodowska Curie actions 2014 • PhD fellowship, Fundação para a Ciência e Tecnologia SFRH/BD/79081/2011 2012 • Research assistant fellowship: “Autism Genome Project Consortium” 2011 • CIBEME’2010 awards for personal communication – Research in Health Sciences 2010
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APPENDIX
List of publications
1. Oliveira B*, Mitjans M*, Nitsche MA, Kuo M-F and Ehrenreich H. Excitation-inhibition dysbalance as predictor of autistic phenotypes. Under revision 2. Oliveira B & Ehrenreich H. Pursuing functional connectivity in NMDAR1 autoantibody carriers. Letter to Lancet Psychiatry. 2018; 51:21-22 3. Pan H*, Oliveira B*, Dere E, Tapken D, Mitjans M, Seidel J, Wesolowski J, Wakhloo D, Klein-Schmidt C, Ronnenberg A et al. Uncoupling the widespread occurrence of anti- NMDAR1 autoantibodies from neuropsychiatric disease in a novel autoimmune model. Molecular Psychiatry. 2018; In press 4. Mitjans M, Begemann M, Ju A, Dere E, Wüstefeld L, Hofer S, Hassouna I, Balkenhol J, Oliveira B, Van der Auwera S et al. Sexual dimorphism of AMBRA1 related autistic features in human and mouse. Translational Psychiatry. 2017; 710:e1247 5. Loureiro S, Almeida J, Café C, Conceição I, Mouga S, Beleza A, Oliveira B, de Sá J, Carreira I, Saraiva J et al. Copy number variations in chromosome 16p13.11-The neurodevelopmental clinical spectrum. Current Paediatric Research. 2017; 21 1: 116- 129 6. Conceição IC, Rama MM, Oliveira B, Café C, Almeida J, Mouga S, Duque F, Oliveira G, Vicente AM. Definition of a putative pathological region in PARK2 associated with autism spectrum disorder through in silico analysis of its functional structure. Psychiatry Genetics. 2017; 272:54-61 7. Martins S, Maia N, Loureiro J, Oliveira B, Marques I, Santos R, Jorge P. Contraction of fully-expanded FMR1 alleles to the normal range: predisposing haplotype or rare events? Journal of Human Genetics. 2016; 622:269-275 8. Castillo-Gómez E*, Oliveira B*, Tapken D*, Bertrand S, Klein-Schmidt C, Pan H, Zafeiriou P, Steiner J, Jurek B, Trippe R et al. All naturally occurring autoantibodies against the NMDA receptor subunit NR1 have pathogenic potential irrespective of epitope and immunoglobulin class. Molecular Psychiatry. 2017; 2212:1776-1784. 9. Ehrenreich H, Castillo-Gomez E, Oliveira B, Ott C, Steiner J, Weissenborn K. Circulating NMDAR1 autoantibodies of different immunoglobulin classes modulates evolution of lesion size in acute ischemic stroke. Neurology Psychiatry and Brain Research. 2016; 221. 10. Ott C, Martens H, Hassouna I, Oliveira B, Erck C, Zafeiriou M-P, Peteri U-K, Hesse D, Gerhart S, Altas B et al. Widespread expression of erythropoietin receptor in brain and its induction by injury. Molecular Medicine. 2015; 21:803-815. 11. Hadley D, Wu Z-l, Kao C, Kini A, Mohamed-Hadley A, Thomas K, Vazquez L, Qiu H, Mentch F, Pellegrino R, Kim C, Connolly J, AGP Consortium et al. The impact of the
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metabotropic glutamate receptor and other gene family interaction networks on autism. Nature Communications. 2014; 5:4074. 12. Pinto D, Delaby E, Merico D, Barbosa M, Merikangas A, Klei L, Thiruvahindrapuram B, Xu X, Ziman R, Wang Z, … Oliveira B et al. Convergence of genes and Cellular Pathways dysregulated in Autism Spectrum Disorders. American Journal Human Genetics. 2014; 5:677-94. 13. Correia CT, Conceição IC, Oliveira B, Coelho J, Sousa I, Sequeira AF, Almeida J, Café C, Duque F, Mouga S et al. Recurrent duplications of the Annexin A1 gene ANXA1 in autism spectrum disorders. Molecular Autism. 2014; 10;51:28. 14. Jorge P*, Oliveira B*, Marques I, Santos R. Development and validation of a multiplex- PCR assay for X-linked intellectual disability. BMC Medical Genetics. 2013; 14:80. 15. Leblond CS, Heinrich J, Delorme R, Proepper C, Betancur C, Huguet G, Konyukh M, Chaste P, Ey E, Rastam M, ... Oliveira B et al. Genetic and functional analyses of SHANK2 mutations provide evidence for a multiple hit model of autism spectrum disorders. PLoS Genetics. 2012; 82: e1002521.
*Authors with equal contribution
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